WO2008029191A1 - Extraits aqueux d'eugenia et leur application pour traiter des troubles associés au diabète - Google Patents

Extraits aqueux d'eugenia et leur application pour traiter des troubles associés au diabète Download PDF

Info

Publication number
WO2008029191A1
WO2008029191A1 PCT/IB2006/002732 IB2006002732W WO2008029191A1 WO 2008029191 A1 WO2008029191 A1 WO 2008029191A1 IB 2006002732 W IB2006002732 W IB 2006002732W WO 2008029191 A1 WO2008029191 A1 WO 2008029191A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
extract
eugenia
extracts
diabetes
Prior art date
Application number
PCT/IB2006/002732
Other languages
English (en)
Inventor
Patell Villoo Morawala
Original Assignee
Avestha Gengraine Technologies Pvt. Ltd
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
Application filed by Avestha Gengraine Technologies Pvt. Ltd filed Critical Avestha Gengraine Technologies Pvt. Ltd
Priority to PCT/IB2006/002732 priority Critical patent/WO2008029191A1/fr
Publication of WO2008029191A1 publication Critical patent/WO2008029191A1/fr

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)
    • A61K36/61—Myrtaceae (Myrtle family), e.g. teatree or eucalyptus

Definitions

  • the present invention relates to herbal extracts from Eugenia jambolana species with hypoglycemic activity, which is characterized in delaying of onset and / or management of diabetes by inhibiting the glucose absorption in the intestine and by mimicking and potentiating the insulin activity and also to a method for producing the extract and the use of the extract in the treatment of diabetes.
  • the invention relates to the extraction process of derivation of the extract from Eugenia jambolana, which is characterized in that the solvent used is water.
  • the herbal extracts comprise active ingredients that delay saccharide digestion or absorption thereby suppressing a rise in postprandial and / or fasting blood glucose levels.
  • IDDM insulin-dependent diabetes mellitus
  • NIDDM non-insulin-dependent diabetes mellitus
  • IDDM results from insulin deficiency caused by cell-mediated autoimmune destruction of pancreatic beta cells, and generally develops in the young (Bach J F., Insulin-dependent diabetes mellitus as a beta cell targeted disease of immunoregulation. J. Autoimm. 8:439-463,1995).
  • IDDM accounts for approximately 10-15% of the diabetic population worldwide (World Health Organization Study Group. Diabetes mellitus. WHO Tech. Rep. Ser. 727:1-113,1985). In contrast, NIDDM results from a variable combination of insulin resistance and insulin deficiency and generally develops in adults (Jun H S, et al., Pathogenesis of non-insulin- dependent (Type II) diabetes mellitus (NIDDM) Genetic predisposition and metabolic abnormalities. Advanced Drug Delivery Reviews 35:157-177, 1999; DeFronzo R A., The triumvirate beta cell, muscle, liver: a collusion responsible for NIDDM. Diabetes 37:667- 687, 1988).
  • NIDDM can also develop at a younger age, as seen in the maturity- onset diabetes of the young (Pirart J., Diabetes mellitus and its degenerative complications: a prospective study of 4400 patients observed between 1947 and 1973. Diabetes Care 1:168-188, 1978). NIDDM accounts for over 85% of the diabetic population worldwide.
  • Factors responsible for causing diabetes are heredity and obesity.
  • Heredity increases the susceptibility of beta cells to viral invasions or favor the development of autoimmune antibodies against the beta cells, thus leading to their destruction.
  • Obesity decreases the number of insulin receptors in the insulin target cells throughout the body. Hence, the amount of insulin present is inadequate to induce its usual metabolic effects.
  • blood glucose levels goes as high as 1200 mg/dl are known to occur which is 12 times higher than the normal. Levels of 300 mg/dl to 500 mg/dl are common in diabetic patients.
  • Diabetic Symptoms which may arise due to pathological physiology of Insulin lack, are Polyuria, Polydipsia, Polyphagia, Asthenia and diminished utilization of carbohydrates for energy.
  • Diabetic Neuropathy is the most common and affects patients at earlier stages.
  • the usual methods for diagnosing diabetes are based on various chemical tests of urine and the blood viz, Urinary Sugar, Fasting Blood Glucose Level, Postprandial Blood Glucose Level (Glucose Tolerance Test) and Acetone breath.
  • Reduction of carbohydrate absorption in the intestine of animals, especially humans is nutritionally and medically of great importance. Reduction of absorption can for example facilitate body weight management, e.g. as part of a method of treating obesity, and can be advantageous for subjects suffering form diabetes or hypoglycemic state.
  • Jamun seeds are widely used in Indian folk medicine for the treatment of diabetes mellitus (Prince P. S., Menon V. P. and Pari L. J Ethnopharmacol; 1998 May; 61 (1): 1-7). It has been reported that the aqueous extract of Jamun seeds has hypoglycemic action.
  • Eugenia jambolana or Syzygium cumini L belongs to the myrtaceae plant family. Common names are Java plum, black plum, jambul and Indian blackberry. It grows naturally is clayey loam soil in tropical as well as sub-tropical zones. It is widely cultivated in Haryana as well as the rest of the Indo-Gangetic plains on a large scale. Its habitat starts from Sri and extends up to Afghanistan. It is generally cultivated as a roadside avenue tree as well.
  • water has been used as solvent to obtain extracts from the plant of genus Eugenia and these extracts have been biologically screened for their potential in the treatment / management of diabetes related disorders.
  • the subject matter of the current invention describes extracts isolated from Eugenia jambolana specifically using water as a solvent which was the result of the planned experiments conducted to test the hypoglycemic potential of the said extracts, further analysis of their potential positive hypoglycemic properties which include its potent inhibitory effect on glucose absorption in the intestine.
  • Figure 1 Shows the free radical scavenging ability of the Eugenia water based extract comparable to ascorbic acid standard.
  • Figure 2 Shows the percent polyphenol content of the Eugenia water based extract comparable to gallic acid standard.
  • Figure 3 Shows the IC50 ( ⁇ g/ml) value for a-glucosidase (0.2 EU) inhibition of the Eugenia water based extract (42.25 ⁇ g/ml) compared to acarbose standard in a reaction mixture containing 3mM Para-nitro-phenyl-D-glucose.
  • Figure 4 Shows Insulin mimetic activity of the 3.33 ⁇ g of Eugenia water based extract compared to that of 1OnM Insulin using tritiated glucose as an energy source in 3T3 L-I adipocyte cell lines.
  • Figure 5 Shows Insulin sensitization activity of the 3.33 ⁇ g of Eugenia water based extract compared to that of 1OnM Insulin using tritiated glucose as an energy source in 3T3 L-I adipocyte cell lines.
  • Figure 6 Shows Insulin mimetic activity of the 3.33 ⁇ g of Eugenia water based extract compared to that of 1OnM Insulin using tritiated glucose as an energy source in C2C12 myocyte cell lines.
  • Figure 7 Shows Insulin sensitization activity of the 3.33 ⁇ g Eugenia water based extract compared to that of 1OnM Insulin using tritiated glucose as an energy source in C2C12 myocyte cell lines.
  • Figure 8 Shows the percent changes in fasting blood glucose level of diabetic Sprague dawley rats at different levels of Eugenia water based extract intervention (50, 125 and 250 mpk) compared to that of baseline values over 5 weeks.
  • Figure 9 Shows the percent changes in post-prandial blood glucose level of diabetic Sprague dawley rats at different levels of Eugenia water extract intervention (50, 125 and 250 mpk) compared to that of baseline values over 5 weeks.
  • the primary objective of the present invention is to identify, test, characterize and screen extracts isolated from the plant species of family Myrtaceae, preferably of the genus Eugenia for their potent inhibitory effects on diabetes.
  • This invention is a further description of the extracts from Eugenia jambolana, their uses and methods for preparation.
  • Another object of the invention is to provide a method of treating diabetes comprising administering to a person in need thereof an anti- diabetic or hypoglycemic effective amount of a composition comprising the extracts of the plant Eugenia jambolana.
  • a particular embodiment of the invention describes the method of a suitable aqueous solvent-based extraction of a specific therapeutically important phytochemical extract.
  • the solvent used for the extraction process is water.
  • the therapeutic potential of the said extracts has been studied and confirmed through standard in vitro cell free and cell based assays.
  • Plant material suitable for preparation of the plant extract for inclusion of the therapeutic composition of the invention is derived from a potential plant administered to a person suffering from diabetes, which results in the lowering of the blood glucose level of the patient. Administration of the composition to the patient both prevented and treated incidences of clinical diabetes.
  • the potential plant is a member of the family Myrtaceae.
  • the potential plant is a member of the genus Eugenia. It will be readily apparent to one skilled in art that other extracts capable of potential positive anti-diabetic properties could be isolated using similar techniques from a wide range of plants i.e., potential plants.
  • the potential plants include all species of the family Myrtaceae, including terrestrial, aquatic or other plants that can be subjected to standard extraction procedures such as those described herein in order to generate an extract that can be tested for its therapeutic abilities.
  • the present invention is directed to a herbal medicinal composition comprising the foregoing plant extracts that can be administered to a personal suffering from diabetes which results in the lowering of the blood glucose level of the patient.
  • anti-diabetic or “hypoglycemic” compound or composition generally refers to an agent that lowers blood glucose levels. If blood glucose level is decreased by at least about 100 mg/dl, then the compound is considered to be a hypoglycemic agent.
  • the hypoglycemic or anti-diabetic effect can be measured by a variety of methods including, but not limited to, measuring the blood glucose levels, the rate of insulin binding to its receptor, the level of insulin secretion from pancreatic beta cells, and inhibition of glucohydrolase activity.
  • extract refers to a concentrated preparation of the essential constituents of the medicinal plant. Typically, drying and powderizing the plant prepare an extract. Optionally, the plant, the dried plant or the powderized plant may be boiled in solution. The extract may be used in liquid form, or it may be mixed with other liquid or solid medicinal herbal extracts. Alternatively, the medicinal herbal extract may be obtained by further precipitating solid extracts from the liquid form.
  • Anti-diabetic means the activity useful for the "treatment” of "diabetes", which includes the prevention of the development of diabetes, and/or the treatment of established diabetes; it also includes the prevention of the causes of diabetes, and/or the decrease or disappearance of its symptoms and/or consequences.
  • a process for obtaining a plant extract possessing hypoglycemic properties comprising (a) obtaining plant material from one or more plants (b) obtaining an extract from the plant material by contacting the plant material with an aqueous solvent such as water thereby providing one or more plant extracts (c) analyzing the plant extracts for free radical scavenging potential, Intestinal alpha-glucosidase inhibition potential, in- vitro screening of Eugenia water based extracts for glucose uptake and the in- vivo efficacy studies.
  • the plant material employed in the extraction process can be the entire potential plant, or it can be one or more distinct tissues from the plant for example, leaves, seeds, roots, stems, flowers, or various combinations thereof but preferably the seed of the plant.
  • the plant material can be treated prior to extraction, for example, by drying, freezing, lyophilizing, or some combination thereof.
  • the plant material can be fragmented and/or homogenized by some means such that a greater surface area is presented to the solvent.
  • the plant material can be crushed or sliced mechanically, using a grinder or other device to fragment the plant parts into small pieces or particles, or the plant material can be frozen liquid nitrogen and then crushed or fragmented into smaller pieces.
  • the solvent used for the extraction process can be aqueous, alcoholic or organic, or a combination thereof.
  • plant material is extracted with an aqueous solvent.
  • suitable solvents include but are not limited to water, buffers, cell media, dilute acids or bases and the like.
  • the plant material is extracted with an alcoholic solvent.
  • suitable alcoholic solvents include, but are not limited to methanol, ethanol, n-propanol, iso-propanol, 2-butanol, ter-butanol, and combinations thereof.
  • water is used as solvent for extraction purposes.
  • the coarse powder of herbal material is soaked in water for a fixed duration at room temperature with constant stirring in pharmaceutical grade solvent extractor.
  • the extract is passed though a continuous centrifuge rotating at 3000rpm to separate extract supernatant and material debris.
  • the clear supernatant obtained is concentrated to 1/10 volume using concentrator and is treated with food grade organic solvents in a definite proportion to remove any unpleasant smell, taste and color.
  • the concentrated and organoleptically optimized extract is dried completely using either spray drier or vacuum drier to get powdered extract.
  • Thus prepared dried extract is stored in airtight food grade plastic bins and the same is taken through several in-vitro cells free and cell based bioassay to validate the extract efficacy.
  • the extract is generally produced by contacting the solid plant material with a solvent with adequate mixing and for a period of time sufficient to ensure adequate exposure of the solid plant material to the solvent such that inhibitory activity present in the plant material can be taken up by the solvent.
  • the solvent extraction process may be selected from direct and successive extraction types such as extraction from plant parts in soxhlet apparatus or in flasks at room temperature or at higher temperature with polar and/or non-polar solvent (s). Regardless of the number of extraction processes, each extraction process typically is conducted over a period of time between about 6 hours to 24 hours at room temperature. Shaking the suspension can encourage adequate contact of the solvent with the plant material.
  • the liquid fraction is then separated from the solid (insoluble) matter resulting in the generation of two fractions: a liquid fraction, which is the potential extract, and a solid fraction. Separation of the liquid and solid fractions can be achieved by one or more standard processes known to those skilled in art.
  • the present invention contemplates the large-scale preparation of the selected plant extracts of the invention.
  • Such extracts can be prepared on a commercial scale by repeating the extraction process that lead to the isolation of the extract of interest.
  • the small-scale extraction procedure can simply be scaled up and additional steps of quality control can be included to ensure reproducible results for the resulting extracts.
  • modifications to the small-scale procedure that may be required during the scale up for the industrial level production of the extract.
  • modifications may include for example, alterations to the solvent being used or to the extraction procedure per se employed in order to compensate for variations that occur during the scale-up and render the overall procedure more amenable to industrial scale production, or more cost effective. Modifications of this type are standard in the industry and would be readily apparent to those skilled in the art.
  • concentration of the purified extracts or partially purified extracts by solvent removal from the original extract and/or fractionated extract, and/or purified extract.
  • solvent removal include, but are not limited to rotary evaporation, distillation (normal and reduced pressure), centrifugal vacuum evaporation (speed vac), and lyophilisation.
  • the potential extracts obtained thereof are concentrated and solubilised in an appropriate solvent preferably ethyl acetate.
  • an appropriate solvent preferably ethyl acetate.
  • organic solvents include but are not limited to, di-ethyl ether, hexane, heptane, dichloromethane, ethyl acetate, butyl alcohol, ether, acetone and the combinations thereof.
  • Oxidative stress is a general term used to describe a state of potential oxidative damage caused by free radicals. Increasing evidence suggests that increased oxidative stress and changes in nitric oxide (NO) formation or activity play a major role in the complications of diabetes.
  • Free radical reactions and non-enzymatic glycosylation may play important roles not only in the development of diabetes but also in its complications.
  • Hayakawa et al Free radicals and diabetes mellirus, Nippon Ronen Igakkai Zasshi. 1990 Mar; 27(2): 149-54.
  • the isolated extracts were used to estimate its free radical scavenging potency relative to ascorbic acid by using Calorimetric-DPPH method (Polterait O. (1997) Anti Oxidants and free-radical Scavengers of Natural origin Current Org. Chem. 1. 415-440).
  • the Eugenia water extract isolated from the seeds of the plant showed nearly 53% of free radical scavenging potency equivalent to that of ascorbic acid.
  • apple polyphenols have a positive effect on diabetes and insulin resistance in animals and humans.
  • European researchers announced that an apple-derived polyphenol, phlorizin, "completely normalized insulin response" in diabetic rats.
  • scientists at the National Institutes of Health in the U.S. gave this same apple polyphenol to mice.
  • Two weeks of treatment "significantly decreased blood glucose levels” in diabetic mice.
  • Whole body fat mass was also “significantly reduced.”
  • the total polyphenol content of the Eugenia water based extract was estimated relative to gallic acid using Calorimetric - Singleton method (Singleton, V.L. and Ropssi, J. A. Jr (1965). Calorimetry of total phenolics with phosphomolybdic — phosphotungstic acid reagent, Am. J. Enol. Vitig. 16: 144 — 158).
  • the Eugenia water extract showed 46 ⁇ 2.25 % total phenol content equivalent to gallic acid clearly indicative of the potential beneficial effects the extracts possess with respect to the management of diabetes and its medicative properties.
  • oligosacharrides (carbohydrates having 2 to 10 glucose residues connected by 1-4 or 1-6 ⁇ -D-glycosidic linkage) into monosaccharides by alpha- glucohydrolase-catalyzed enzymatic reactions was tested for Eugenia water extract using Calorimetric - para-nitro-phenyl (pNP) release method using pNP-a-D-glucoside (Halvorson. H, 'Methods in enzymology' VoI 8, Academics Press, New York, 1966, p 559-562).
  • the Eugenia water extract showed greater ⁇ -glucosidase inhibition potential (IC50 value of 42.25 ⁇ g/ml) relative to the commercially available ⁇ - glucosidase inhibitor, acarbose (IC50 value of 146.55 ⁇ g/ml) for 0.2 ⁇ -glucosidase enzyme units at standard enzymatic reaction conditions.
  • IC50 value 42.25 ⁇ g/ml
  • acarbose IC50 value of 146.55 ⁇ g/ml
  • Insulin-stimulated glucose uptake in adipose tissue and striated muscle is critical for reducing postprandial blood glucose concentration and the dysregulation of this process is one of the hallmarks of Type -II Diabetes mellitus (Non Insulin dependent). Oral therapies for Diabetes mellitus have emerged out of this interest and are widely used still today. But rather than acting by mimicking insulin, these drugs acts either by stimulating insulin release [Sulphonylurease], potentiating insulin action (thiazolidinedione) or lowering hepatic glucose production (biguanides). Various amounts of Eugenia water extract (0.034 ⁇ g to 33.4 ⁇ g) are tested for insulin mimetic and sensitization effects with / without insulin.
  • Radio labeled glucose is used to measure the changes in the level of glucose uptake activity of the adipocyte cells in response to treatment with samples in the presence or absence of insulin.
  • the assay is performed in a 96-well microtiter plate format and the counts per minute are measured using a radioactive counter. The count per minute can be measured on a microtitre plate by radioactive counter.
  • the present invention envisages the method of treating diabetes and other related diseases thereof by administering an effective amount of the therapeutic composition comprising the single plant extract or the screened plant extracts purified there from in combination.
  • the therapeutic compositions of the invention can be administered alone or in combination with one or more standard anti-diabetic therapeutics.
  • the present invention also contemplates the administration of sub-optimal doses of the therapeutic composition, for example, chemotherapeutic drug(s), in combination with the therapeutic composition.
  • one or more plant extracts in order to prepare a therapeutic combination, one or more plant extracts is first selected and then the efficacy of the extract(s) in controlling diabetes and maintaining glucose homeostasis is determined using standard techniques as one of those outlined above.
  • the efficacy of the one or more plant extract alone is then compared to the efficacy of the one or more plant extract in combination with varying amounts of another component i.e., another plant extract.
  • the invention also contemplates the combination the plant extract with another synthetic inhibitors controlling blood glucose levels. A combination that demonstrates therapeutic index in comparison to the individual properties is considered to be an effective combination.
  • compositions comprising two or more plant extracts various ratios of the constituent plant extracts are contemplated.
  • the ratio of extract A to extract B can vary anywhere between 1:99 and 99:1.
  • anywhere between 99:1 and 1:99 it is meant that the ratio of the two extracts can be defined by any ratio within this ratio can be between 98:2 and about 1 :99 between about 98:2 and 2:98, between 97:3 and 1:99, between 97:3 and 2:98, between 97:3 and 3:97, etc.
  • the present invention contemplates the ratio of the two extracts is between about 90:10 and 10:90, 80:20 and 20:80, 70:30 and 30:70, 60:40 and 40:60 or 50: 50.Analogous ratios are contemplated for compositions comprising more than two or more plant extracts.
  • the formulations of the present invention contain at least an effective amount of the therapeutic composition.
  • the effective amount is considered to be that amount of the composition, in weight percent of the overall formulation, which must be present in order to produce the desired therapeutic effect.
  • the effective amount may vary, depending upon, for example the disease to be treated and the form of administration.
  • the therapeutic composition will be present in an amount ranging from about 1% to 100% by weight of the formulation, 10% to about 90% by weight of the formulation, 20% to about 80% by weight of the formulation, 30% to 70% by weight of the formulation, from about 40% to 60% by weight of the formulation and about 50% by weight of the formulation.
  • the present invention contemplates the use of the therapeutic compositions at various stages in the disease development and progression, including in the treatment of early stage, or advanced and/or aggressive stage of hyperglycemia, diabetes or related disorders.
  • the administration of the therapeutic composition comprising the isolated and screened extracts to mammal having an early stage of the disorder can help to attenuate the progression of the disease.
  • the dosage of the therapeutic composition to be administered is not subject to defined limits, but will usually be an effective amount. However it will be understood that the actual amount of the composition to be administered will be determined by a physician, in the light of the relevant circumstances, including the exact condition to be treated, the chosen route of administration, the actual composition administered, the age, the weight, and the response of the individual patient and the severity of the patient's symptoms.
  • the dosage ranges are not intended to limit the scope of the invention in any way.
  • the medical composition of the present invention comprising a carbohydrate inhibitor as an active ingredient can be effectively used as an anti-diabetic medicine due to its effect of suppressing a postprandial rise and fasting levels in blood glucose levels (hyperglycemia) by. delaying I digestion and absorption of saccharides in vivo (intestinal) .
  • the anti-diabetic medicine broadly encompasses those that can prevent or improve diabetes.
  • the anti-diabetic medicine of the present invention includes those that can prevent an onset of diabetes in a subject (including humans and other animals) who has the potential of suffering from an onset of diabetes, due to its effect for suppressing a postprandial and / or fasting rise in blood glucose levels.
  • the anti-diabetic medicine of the present invention encompasses those that have an effect for ameliorating the hyperglycemic condition of a subject (including humans and other animals).
  • the anti diabetic medicine of the present invention also encompasses those that have an effect for preventing or ameliorating diseases attributable to hyperglycemia, such as diabetic complications, by suppressing or ameliorating blood glucose levels (reducing the blood glucose level from hyperglycemic condition).
  • the diabetes targeted by the present invention is preferably type II diabetes.
  • the present water based plant extracts can also be used for the management of other diabetic complications which are general or local diseases directly or indirectly caused by diabetes. Specific examples thereof are diabetic acidosis, diabetic xanthoma, diabetic myatrophy, diabetic ketosis, diabetic coma, diabetic stomach disorders, diabetic gangrene, diabetic ulcer, diabetic diarrhea, diabetic microangiopathy, diabetic uterosclerosis, diabetic cardiomyopathy, diabetic neuropathy, diabetic nephropathy, diabetic blister, diabetic cataract, diabetic dermatitis, diabetic scleredema, diabetic retinopathy, diabetic necrobiosis lipoidica, diabetic blood flow obstructions, etc.
  • the administration amount of the medical composition are not limited, and can be suitably selected depending on the targeted treatment effects, administration method, treatment period, sex and / or age of the subject, etc.
  • the therapeutic compositions comprising the plant extract are not limited to only those for humans but also include those for various animals, in particular, other mammals. Therefore, the food compositions include foods for animals such as cats, dogs, and the like pets, and the medical compositions include those for animals other than humans.
  • the therapeutic composition can be formulated as a pharmaceutical or naturopathic formulation such as phytoceuticals or nutraceuticals, for oral, topical, rectal or parenteral administration or for administration by inhalation or spray.
  • the phytoceutical or naturopathic formulation may comprise the one or more plant extracts in dosage unit formulations containing the conventional non-toxic physiologically acceptable carriers, adjuvants and vehicles.
  • the pharmaceutical or naturopathic formulations may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion hard or soft capsules, or syrups or elixirs.
  • the therapeutic compositions of the invention may be formulated as phytoceuticals, or nutraceuticals.
  • Phytoceuticals may optionally comprise other plant-derived components and can therefore be delivered by such non-limiting vehicles as teas, tonics, juices or syrups.
  • Nutraceuticals contemplated by the present invention may provide nutritional and/or supplemental benefits and therefore be delivered, for example as foods, dietary supplements, extracts, beverages or the like.
  • Phytoceutical and nutraceuticals can be administered in accordance with conventional treatment programs and/or may be a part of the dietary or supplemental program.
  • Formulations intended for oral use may be prepared according to methods known in art for the manufacture of pharmaceutical compositions and may contain one or more agents selected from the group of flavoring agents, coloring agents and preserving agents in order to provide palatable preparations.
  • Tablets contain the active ingredient in admixture with suitable non-toxic physiologically acceptable excipients including, for example, inert diluents, such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch, or alginic acid, binding agents, such as starch, gelatine or acacia, and lubricating agents, such as magnesium stearate, stearic acid or talc.
  • suitable non-toxic physiologically acceptable excipients including, for example, inert diluents, such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch, or alginic acid, binding agents, such as starch, gelatine or acacia, and lubricating agents, such as magnesium stearate, stearic acid or talc.
  • suitable non-toxic physiologically acceptable excipients including, for example,
  • additives or carriers can be incorporated into the orally delivered pharmaceutical naturopathic formulations or the invention.
  • Optional additives of the present composition include, without limitation, phospholipids, such as phosphatidyl glycerol, phosphotidyl inositol, phosphotidyl serine, phosphotidyl choline, phosphotidyl ethanolamine as well as phosphatidic acids, ceramide, cerebrosides, sphingomyelins and cardiolipins.
  • compositions for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatine capsules wherein the active ingredient is mixed with water or an oil based medium such as peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • water or an oil based medium such as peanut oil, liquid paraffin or olive oil.
  • Oily suspensions may be formulated by suspending the plant extract(s) in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Flavoring agents may be added to provide palatable oral preparations. These formulations can be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation suitable for an aqueous suspension by the addition of water provide the active ingredient in admixture with dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents, sweetening, flavoring and coloring agents may also be present.
  • a comestible that is to say, a foodstuff comprising at least an extract of the invention, typically in dried form, such as in a lyophilised form.
  • cosmetibles may contain more than one extract of the invention and may be used.
  • Such foodstuffs may be used in a prophylactic manner and may contain further extracts having a similar function to the first added extract or further added extracts may be added that have a different prophylactic function.
  • a foodstuff could either comprise extracts that provide for a comestible having a single functional aspect, or a comestible may have a multi-functional prophylactic effect against two or more disease types.
  • the type of foodstuff or comestible to which at least an extract of the invention may be added includes any processed food such as confectionaries, baked products including breads such as loafs, and flat breads such as pitta bread, naan bread and the like, cakes, snack foods such as museli bars, compressed dried fruit bars, biscuits, dairy products such as yoghurts, milk and milk-based products such as custards, cream, cheese, butter and creme fraiche, simulated dairy food product such as Elmlea products, fruits and vegetable juices, aerated drinks, such as carbonated soft drinks and non-aerated drinks such as squashes, soya milk, rice milk and coconut milk and the like, pastas, noodles, vegetables, seed and nut oils, fruited oils such as sunflower oil, rapeseed oil, olive oil, walnut, hazelnut, and sesame seed oil and the like, and frozen confectionaries such as ice cream, iced yoghurts and the like.
  • processed food such as confectionaries,
  • the water based extract derived from Eugenia jambolana is safe and can be effective for reducing and preventing obesity by suppressing digestion and absorption of saccharides from the alimentary canal. Furthermore the postprandial and the fasting blood glucose levels can be considerably reduced thereby can be effectively used for ameliorating diabetic hyperglycemic conditions and preventing development in disorders of a diabetic patient caused by hyperglycemia.
  • the present invention relates to mixtures, which can be isolated from Eugenia jambolana, L seeds (Myrtaceae family) for the management of an important clinical problem like diabetes.
  • the preparation method is exemplified as follows:
  • Eugenia jambolana seeds are selected and tested for seed quality by pharmacognosy techniques.
  • the authentic plant material is procured for bioactive extraction.
  • Good quality Eugenia jambolana seeds are macerated into solvent extractable seed powder of preferable size of lOO ⁇ m.
  • the bioactive extract is prepared from the seeds.
  • Eugenia jambolana water mixtures are used as solvent for the preparation of the bioactive extract.
  • the extraction process is carried out in a solvent extraction vessel of 100-liter capacity. The extraction process is undertaken at a temperature of 25 0 C and for a duration of 240 minutes. The extraction is carried out with constant stirring at the rate of 200 rpm allowing the heavier particles in the extraction mixture to settle down and later the mixture is allowed to stand undisturbed for 120 minutes.
  • the liquid phase of the extract is clarified by centrifugation.
  • the centrifugation process is undertaken at 3000 rpm and at room temperature.
  • the clarified extract was then concentrated to 1/10 of the total volume.
  • the concentrated extract (two parts) is mixed well with a moderately non- polar solvent such as ethyl acetate (one part) in an extraction vessel for 60 minutes and left undisturbed for 120 minutes.
  • the lower water phase is then separated and dried completely to powder using either spray drier or vaccum dried under reduced pressure.
  • the powdered extract is then stored in an airtight plastic container. This extract is further used for all the biochemical, physiochemical, heavy metal, natural toxin and detailed efficacy study.
  • the total polyphenol content of the Eugenia water extract is estimated relative to gallic acid using Calorimetric - Singleton method (Singleton, V.L. and Ropssi, J. A. Jr (1965). Calorimetry of total phenolics with phosphomolybdic - phosphotungstic acid reagent, Am. J. Enol. Vitig. 16: 144 - 158).
  • the Eugenia water based extract showed 46 ⁇ 2.25 % total phenol content equivalent to gallic acid, which is shown in fig 2.
  • oligosacharrides carbohydrates having 2 to 10 glucose residues connected by 1-4 or 1-6 alpha-D-glycosidic linkage
  • pNP Calorimetric - para-nitro-phenyl
  • the Eugenia water based extract showed greater ⁇ -glucosidase inhibition potential (IC50 value of 42.25 ⁇ g/ml) relative to the commercially available - ⁇ glucosidase inhibitor, acarbose (IC50 value of 146.55 ⁇ g/ml) for 0.2 ⁇ -glucosidase enzyme units at standard enzymatic reaction conditions.
  • IC50 value 42.25 ⁇ g/ml
  • acarbose IC50 value of 146.55 ⁇ g/ml
  • Example 5 In- vitro screening of Eugenia water based extract for glucose uptake
  • Insulin-stimulated glucose uptake in adipose tissue and striated muscle is critical for reducing postprandial blood glucose concentration and the dysregulation of this process is one of the hallmarks of Type —II Diabetes mellitus (Non Insulin dependent). Oral therapies for Diabetes mellitus have emerged out of this interest and are widely used still today. But rather than acting by mimicking insulin, these drugs acts either by stimulating insulin release [Sulphonylurease], potentiating insulin action (thiazolidinedione) or lowering hepatic glucose production (biguanides). Various amounts of Eugenia water extracts (0.034 ⁇ g to 33.4 ⁇ g) are tested for insulin mimetic and sensitization effects with / without insulin.
  • Radio labeled glucose is used to measure the changes in the level of glucose uptake activity of the adipocyte cells in response to treatment with samples in the presence or absence of insulin.
  • the assay is performed in a 96-well microtiter plate format and the counts per minute are measured using a radioactive counter. The count per minute can be measured on a microtitre plate by radioactive counter.
  • the amount that showed best insulin mimetic and sensitization activity in 3T3L-1 adipocyte cells and C2C12 myocyte cells for glucose uptake was around 0.334 ⁇ g.
  • the method followed for screening is as follows:
  • Preadipocytes (3T3L-1) and premyocytes (C2C12) are cultured in DMEM containing 10%FCS, 4mM Glutamine, 2 % NaHCO3 and antimycotic, in an atmosphere of 5% CO2 at 370C, separately.
  • Myoblasts are cultured up to 80% confluency and the cells are sub- cultured at three-day intervals.
  • 20,000 of preadipocytes and myocytes are seeded separately in each well of a 96 well plate and differentiated for 48 hours in DMEM:F12(1:1), 0.5mMIBMX, 0.25mM Dexmethasone and lug Insulin for 48hrs followed by incubation with lug of Insulin for 8hours.
  • the ability of the plant extract to induce glucose uptake is tested in two different ways 1) glucose uptake in presence of insulin (extract + insulin) and 2) glucose uptake in absence of insulin (extract alone). Therefore incubate in duplicate (one set to evaluate glucose uptake in presence of insulin i.e. extract + insulin and other set without insulin i.e. extract alone) with different concentration of extracts (300 ⁇ g/well, 30 ⁇ g/well, 3 ⁇ g/well and 0.3 ⁇ g/well) in triplicates for 18 hours at 370 C and 5%CO2 lOO ⁇ l of DMEM. The medium is then removed and the cells are incubated with KRH buffer (100 microliters) at 370C and 5% CO2 for 10 minutes. Cells are incubated with insulin.
  • KRH buffer 100 microliters
  • the cells are washed three times with ice-cold KRH buffer (lOO ⁇ l). KRH buffer is removed and 20 ⁇ l 1% Triton X is added to each well to lyse the cells and incubate for 10 min at 37 0 C and 5%CO2. 200 ⁇ l of Aqualite is added per well and the supernatant is transferred back to the plates and counted on a micro-titer plate radioactive counter.
  • the results obtained for insulin mimetic and sensitization potential of Eugenia water based extracts are depicted in fig. 4 to fig. 7 using differentiated adipocyte and myocytes. All the observed values of glucose uptake activity are blank corrected using the control (cells alone background value). These values are normalized with MTT cell viability assay values for the corresponding extracts. The degree of insulin mimetic/sensitization activity of each sample concentration is calculated as a percentage of that observed using 1OnM insulin alone.
  • Sprague dawley rats weighing ⁇ 250g with a variation of + 20% of the mean weight are selected for In-vivo efficacy screening of Eugenia water extract.
  • the identification is undertaken by the cage tag and the corresponding picric acid color body markings.
  • the number of animals selected in a group is five and kept in a experimental room after veterinary examination.
  • the route of administration is oral gavage.
  • the rats are fed with 5 % glucose water for two days before STZ injection to avoid death due to hypoglycemic shock. Rats are fasted for 12 hours and injected intraperitoneallly with 45mg/kg-body weight of streptozotocin.
  • Example 7 Screening of Eugenia water extract at 50, 125 and 200 mg/kg-body weight.
  • the dosing formulations are prepared freshly each day 0.5% CMC was used as the vehicle.
  • the test article Eugenia water extract, is administered by oral gavage to each rat daily, for 35 consecutive days.
  • the animals were dosed at approximately the same time each day where possible using a stainless steel intubation needle fitted onto a suitably graduated glass syringe.
  • the dosage volume administered to individual rat was adjusted according to its most recently recorded body weights. Treatment in this manner continued once a day, seven days a week, for a total period of 35 days.
  • Vehicle control group animals are treated with the vehicle only at the same dosage volume i.e. 10 ml/kg body weight.
  • the Groups include: Gl -Vehicle control, G2-Diabetic control, G-3 Diabetic animals treated with Pioglitazone (Standard anti-diabetic drug) 20mg/kg-body weight.
  • G-4 Eugenia water extract 50 mg/kg-body weight
  • G-5 Eugenia water extract 125 mg/kg-body weight
  • G-6 Eugenia water extract 200 mg/kg-body weight.
  • AU signs of ill health, together with any behavioral changes or reaction to treatment are recorded for individual animals. Dated and signed records of appearance, change and disappearance of clinical signs are maintained in clinical history sheets for individual animals.
  • the parameters that are observed or included in the study were postprandial glucose, fasting blood glucose (FBG), body weight and feed consumption. Blood collection by ROP on week 1,2 & 5 for the above said parameters.
  • the animals selected for the study are treated with the extract at 3 different dose levels for 35 days. Necropsy Examination: On completion of 35 days of treatment period, all surviving rats will be sacrificed by exsanguinations under CO2 anesthesia. Complete necropsies were carried out on all animals including those, which died during the study.
  • the postprandial blood glucose lowering potential of Eugenia water extracts at different concentrations are illustrated in figure 9.
  • the diabetic control rat group showed 240% increase in their postprandial blood glucose levels compared to the baseline values of the same group at the start of the study. While the groups treated with pioglitazone, Eugenia water extract at 50, 125 and 200mpk (milligram per kg body weight) did not show any significant percent change from their baseline values. The results have been represented in Table No. 1
  • the fasting blood glucose lowering potential of the Eugenia water extracts at different concentrations are illustrated in figure 8.
  • the glucose lowering response of 125 and 200 mpk Eugenia water extracts are on par with the response of pioglitazone at 20mpk.
  • the results have been represented in Table No. 2.

Landscapes

  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Biotechnology (AREA)
  • Botany (AREA)
  • Medical Informatics (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

La présente invention se rapporte à des extraits végétaux de l'espèce Eugenia Jambolana présentant une activité hypoglycémique, qui sont caractérisés en ce qu'ils retardent l'apparition du diabète et/ou en permettent la gestion thérapeutique, en inhibant l'absorption du glucose dans l'intestin et en imitant et potentialisant l'activité de l'insuline, ainsi qu'à un procédé de production dudit extrait et d'utilisation de ce dernier pour traiter le diabète. L'invention concerne également le processus d'extraction et de dérivation de l'extrait d'Eugenia Jambolana, qui est caractérisé en ce que l'on utilise de l'eau comme solvant. Les extraits végétaux selon l'invention contiennent des ingrédients actifs qui retardent la digestion ou l'absorption des saccharides, ce qui permet d'éviter la hausse des niveaux du glucose sanguin post-prandial et/ou à jeun.
PCT/IB2006/002732 2006-09-08 2006-09-08 Extraits aqueux d'eugenia et leur application pour traiter des troubles associés au diabète WO2008029191A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/IB2006/002732 WO2008029191A1 (fr) 2006-09-08 2006-09-08 Extraits aqueux d'eugenia et leur application pour traiter des troubles associés au diabète

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IB2006/002732 WO2008029191A1 (fr) 2006-09-08 2006-09-08 Extraits aqueux d'eugenia et leur application pour traiter des troubles associés au diabète

Publications (1)

Publication Number Publication Date
WO2008029191A1 true WO2008029191A1 (fr) 2008-03-13

Family

ID=37964574

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2006/002732 WO2008029191A1 (fr) 2006-09-08 2006-09-08 Extraits aqueux d'eugenia et leur application pour traiter des troubles associés au diabète

Country Status (1)

Country Link
WO (1) WO2008029191A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020025349A1 (en) * 2000-05-02 2002-02-28 Brindavanam Narasimha Baba Novel herbal composition for diabetes patients and a process for producing the same

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020025349A1 (en) * 2000-05-02 2002-02-28 Brindavanam Narasimha Baba Novel herbal composition for diabetes patients and a process for producing the same

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ACHREKAR S ET AL: "HYPOGLYCEMIC ACTIVITY OF EUGENIA JAMBOLANA AND FICUS BENGALENSIS: MECHANISM OF ACTION", IN VIVO - INTERNATIONAL JOURNAL OF IN VIVO RESEARCH, XX, GB, vol. 5, no. 2, 1991, pages 143 - 148, XP000608379, ISSN: 0258-851X *
GROVER J K ET AL: "Anti-hyperglycemic effet of Eugenia jambolana and tinospora cordifolia in experimental diabetes and their effects on key metabolic enzymes involved in carbohydrate metabolism", JOURNAL OF ETHNOPHARMACOLOGY, ELSEVIER SCIENTIFIC PUBLISHERS LTD, IE, vol. 73, no. 3, 2000, pages 461 - 470, XP002904902, ISSN: 0378-8741 *
KELKAR S M ET AL: "A SIMPLE TWO-STEP PURIFICATION OF ANTIDIABETIC COMPOUNDS FROM EUGENIA JAMBOLANA FRUIT-PULP: PROTEOLYTIC RESISTANCE AND OTHER PROPERTIES", PHYTOMEDICINE, GUSTAV FISCHER VERLAG, STUTTGART, DE, vol. 3, no. 4, 1996, pages 353 - 359, XP000940531, ISSN: 0944-7113 *
MAHMOUD I I ET AL: "Acylated flavonol glycosides from Eugenia jambolana leaves", PHYTOCHEMISTRY, PERGAMON PRESS, GB, vol. 58, no. 8, December 2001 (2001-12-01), pages 1239 - 1244, XP004328223, ISSN: 0031-9422 *
RAVI K ET AL: "Antihyperlipidemic effect of Eugenia jambolana seed kernel on streptozotocin-induced diabetes in rats", FOOD AND CHEMICAL TOXICOLOGY, XX, XX, vol. 43, no. 9, September 2005 (2005-09-01), pages 1433 - 1439, XP004962082, ISSN: 0278-6915 *
RAVI K ET AL: "Effect of Eugenia Jambolana seed kernel on antioxidant defense system in streptozotocin-induced diabetes in rats", 15 October 2004, LIFE SCIENCES, PERGAMON PRESS, OXFORD, GB, PAGE(S) 2717-2731, ISSN: 0024-3205, XP004568617 *
SHARAM S B ET AL: "Hypoglycaemic and hypolipidemic effect of ethanolic extract of seeds of Eugenia jambolana in alloxan-induced diabetic rabbits", JOURNAL OF ETHNOPHARMACOLOGY, ELSEVIER SCIENTIFIC PUBLISHERS LTD, IE, vol. 85, no. 2-3, April 2003 (2003-04-01), pages 201 - 206, XP002985977, ISSN: 0378-8741 *
SHARMA ET AL: "Antihyperglycemic effect of the fruit-pulp of Eugenia jambolana in experimental diabetes mellitus", JOURNAL OF ETHNOPHARMACOLOGY, ELSEVIER SCIENTIFIC PUBLISHERS LTD, IE, vol. 104, no. 3, 6 April 2006 (2006-04-06), pages 367 - 373, XP005324712, ISSN: 0378-8741 *

Similar Documents

Publication Publication Date Title
KR101412221B1 (ko) 구기자잎 추출 분말과 베타인을 유효성분으로 함유하는 항비만 조성물
US20080003312A1 (en) Method of improving fat metabolism
JP2011032272A (ja) 複合生薬抽出物を有効成分とする糖尿病または糖尿病合併症の治療または予防用組成物
KR101651908B1 (ko) 갈색거저리 유충 또는 이의 추출물을 유효성분으로 포함하는 당뇨 예방 또는 치료용 조성물
US20130178436A1 (en) Composition for preventing, improving, or treating renal disease including maillard browning reaction products of panax species plant extract
MX2007010408A (es) Composiciones que comprenden actinidia y sus metodos de uso.
JP7254727B2 (ja) ハーブ組成物
Zubairu et al. Effect of Anacardium occidentale fruit juice extract on haematological parameters and spleen of paracetamol induced injury in albino rats
US20140148399A1 (en) Compositions and methods for treating or ameliorating obesity or for reducing diabetic hypercholesterolemia
Anaga et al. Antihyperglycaemic properties of the ethyl acetate extract of Dennettia tripetala in diabetic rats
US20110064833A1 (en) Synergistic herbal composition and process of preparation thereof
Belkhodja et al. Phytochemical Screening and Evaluation of the Antiarthritic Potentialof Ammoides pusilla Aqueous Extract on Freund's Adjuvant-Induced Rheumatoid Arthritis
EP3275457A1 (fr) Composition pharmaceutique contenant de la silybine
WO2008059310A1 (fr) Extraits aqueux de cinnamomum zeylanicum et leurs applications pour traiter des troubles associés au diabète
JP2006160710A (ja) 糖尿病の予防・治療用組成物およびその有効成分を含む健康食品
EP2152284A1 (fr) Composition bioactive alimentaire ayurvédique / fonctionnelle synergique [cincata]
WO2018084224A1 (fr) Agent thérapeutique ou prophylactique de la stéatose hépatique non alcoolique et aliment prophylactique de la stéatose hépatique non alcoolique
WO2008041049A1 (fr) Extraits végétaux de cinnamomum zeylanicum destinés au traitement du diabète et procédé d'extraction de celui-ci
US20100129478A1 (en) Plant Extracts and Methods and Uses Therefore
KR101385191B1 (ko) 치커리 추출물의 근육 손상 예방, 치료 또는 개선을 위한 용도
KR101332824B1 (ko) 민대극 추출물을 포함하는 관절염 예방 및 치료용 조성물
WO2008029191A1 (fr) Extraits aqueux d'eugenia et leur application pour traiter des troubles associés au diabète
WO2008136013A1 (fr) Extrait organoleptiquement amélioré de salacia et méthode associée (minora)
WO2008062245A1 (fr) Extraits de la plante eugenia jambolana utilisés dans le traitement du diabète et procédé d'extraction de ceux-ci
KR101508561B1 (ko) 귀리를 포함하는 혼합 생약재 추출물을 함유하는 당뇨병의 예방 또는 치료용 약학조성물

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: 06808920

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: 06808920

Country of ref document: EP

Kind code of ref document: A1