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WO2008062245A1 - Eugenia jambolana plant extracts for the treatment of diabetes and the extraction process thereof - Google Patents

Eugenia jambolana plant extracts for the treatment of diabetes and the extraction process thereof Download PDF

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Publication number
WO2008062245A1
WO2008062245A1 PCT/IB2006/002733 IB2006002733W WO2008062245A1 WO 2008062245 A1 WO2008062245 A1 WO 2008062245A1 IB 2006002733 W IB2006002733 W IB 2006002733W WO 2008062245 A1 WO2008062245 A1 WO 2008062245A1
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plant
extract
eugenia
extracts
diabetes
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PCT/IB2006/002733
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French (fr)
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Patell Villoo Morawala
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Avestha Gengraine Technologies Pvt. Ltd
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Priority to PCT/IB2006/002733 priority Critical patent/WO2008062245A1/en
Publication of WO2008062245A1 publication Critical patent/WO2008062245A1/en

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    • 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.
  • IDDM insulin-dependent diabetes mellitus
  • NDDDM 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).
  • 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 Diabetic Diarrhea
  • Urinary retention Gustatory Swelling
  • Papillary Reflexes Cardiac Autonomic Disturbances
  • Collagen Disturbances Collagen Disturbances.
  • 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.
  • 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 Sy ⁇ ygium 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.
  • Figure 4 Shows Insulin mimetic activity of the 3.33 ⁇ g of Eugenia 70% ethanol 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 Eugenia 70% ethanol 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 70% ethanol extract compared to that of 1OnM Insulin using tritiated glucose as an energy source in C2C12 myocyte cell lines.
  • 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.
  • 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 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 crashed or fragmented into smaller pieces.
  • the coarse powder of herbal material is soaked in 70% ethanol 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. Adequate contact of the solvent with the plant material can be encouraged by shaking the suspension. 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.
  • 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.
  • Free radicals are constantly being generated in the body, as a result of the normal metabolic processes. Under physiological conditions, damage due to free radicals is countered by antioxidants. Sometimes, excessive free radical formation occurs in the body, and the antioxidant systems in the body cannot cope with the situations, i.e., the pro-oxidants overwhelm the antioxidants. This situation is known as oxidative stress. Thus, oxidative stress is a general term used to describe a state of potential oxidative damage caused by free radicals.
  • NO nitric oxide
  • the weight of experimental and human evidence supports a clear role for increased oxidant stress in many of the proposed biochemical pathways linked to the microvascular and microvascular complications of diabetes. In particular, very recent evidence has underscored the particular role the elevated glucose plays in oxidative modification of LDL by a superoxide dependent pathway, and has demonstrated that, in people with poorly controlled IDDM 5 there is increased LDL oxidation associated with reduced antioxidant defenses.
  • 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 mellitus, 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 70% ethanol extract isolated from the seeds of the plant showed nearly 60% 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 70% ethanol 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 70% ethanols extract showed 53.4 ⁇ 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 70% ethanol 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 70% ethanol extract showed greater ⁇ -glucosidase inhibition potential (IC50 value of 32.79 ⁇ 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.
  • 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 rnirnicking insulin, these drugs acts either by stimulating insulin release [Sulphonymrease], potentiating insulin action (thiazolidinedione). or lowering hepatic glucose production (biguanides). Various amounts of Eugenia 70% ethanol 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 inhibitor.
  • 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 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 com 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 com starch, or alginic acid, binding agents, such as starch, gelatine or acacia, and lubricating agents, such as magnesium stearate, stearic acid or talc.
  • the tablets can be uncoated, or they may be coated by
  • 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
  • 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 confectioneries, 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, daily 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 confectionerie
  • 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.70% ethanol 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 70% ethanol 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 70% ethanols extract showed 53.4 + 2.25 % total phenol content equivalent to gallic acid, which is shown in figure 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 70% ethanol extract showed greater ⁇ -glucosidase inhibition potential (IC50 value of 32.79 ⁇ 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.
  • Example 5 Ih- vitro screening of Eugenia 70% ethanol 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 drags acts either by stimulating insulin release [Sulphonylurease], potentiating insulin action (thiazolidinedione) or lowering hepatic glucose production (biguanides).
  • Various amounts of Eugenia 70% ethanol 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 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 4- 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 37 0 C 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°C and 5%CO 2 . 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 70% ethanol extract is depicted in figure 4 to figure 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.
  • Example 6 In- vivo efficacy screening using Sprague dawley rats In-vivo efficacy screening of Eugenia 70% ethanol extract was done using Sprague dawley rats and the procedure undertaken is as follows:
  • Sprague dawley rats weighing ⁇ 250g with a variation of ⁇ 20% of the mean weight are selected for In-vivo efficacy screening of Eugenia 70% ethanol extract.
  • the number of animals per dose group is five and is kept for examination and acclimatization for a week period at the start of the study.
  • the total number of dose groups is six along with vehicle control.
  • the different groups are Gl -Vehicle control, G2-Diabetic control, G-3 Diabetic animals treated with Pioglitazone (Standard anti-diabetic drug).
  • G-4 Eugenia 70% ethanol extract 50 mg/kg-body weight
  • G-5 Eugenia 70% ethanol extract 125 mg/kg-body weight
  • G-6 Eugenia 70% ethanol extract 200 mg/kg-body weight.
  • Blood is collection under CO 2 anesthesia by retro orbital (ROP) plexus method and FBG analysis.
  • Rats are fed with 5 % glucose water for two days before STZ injection and are fasted for 12 hours before adrninistering streptozotocin.
  • the rats are injected intra-peritoneal with 45mg/kg- body weight of streptozotocin in a citrate buffer ph: 4.5 and allowed for week recovery.
  • Rats are fasted for 12 hours after 7 th day of STZ injection and blood collected in heparin as the anti-coagulant, under CO 2 anesthesia and analyzed for FBG. Rats that have the range of FBG beyond 250 mg/dl and above are considered for the study.
  • the in-vivo efficacy screening of Eugenia 70% ethanol extract at different concentrations is started and the dosing is 50, 125 and 200mg per kg body with 0.5 percent of carboxyl-methyl-cellulose (CMC) as vehicle.
  • CMC carboxyl-methyl-cellulose
  • the fasting blood glucose lowering potential of the Eugenia 70% ethanol extracts at different concentrations are illustrated in figure 9.
  • the diabetic control rat group showed 246% increase in their fasting blood glucose levels compared to the baseline values of the same group at the start of the study.
  • the group treated with pioglitazone showed 31.37% increase, while Eugenia 70% ethanol extract at 50, 125 and 200mpk showed 50% increase, 17.6% decrease and 39.36% increase respectively from their baseline values at the end of 35 days.
  • the glucose lowering response of 125 and 200 mpk Eugenia 70% ethanol extracts are on par with the response of pioglitazone at 20mpk. The results have been represented in Table No. 2
  • Diabetic control rats showed 84.35% of HbAIc compared to the control rats (38.19%), pioglitazone treated rats (73.63%) 50m ⁇ k (50.04%) and 125mpk Eugenia 70% ethanol extract treated rats (86.30%).

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Abstract

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.

Description

Eugenia iambolana plant extracts for the treatment of diabetes and the extraction process thereof.
4. FIELD OF THE INVENTION:
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.
BACKGROUND OF THE INVENTION:
Diabetes mellitus is a common, serious disease characterized by hyperglycemia. The- disease can be divided into two major subclasses: insulin-dependent diabetes mellitus (IDDM)5 also known as type I diabetes, and non-insulin-dependent diabetes mellitus (NDDDM), also known as type II diabetes (World Health Organization Study Group. Diabetes mellitus. WHO Tech. Rep. Ser. 727:1-113, 1985). 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). However, 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. In diabetic patients 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. Thus the tendency of diabetes is to cause both extra-cellular and intracellular dehydration to develop (Guyton, Ca & Hall Ej (1996): In Text Book Of Medical Physiology, 9.suρ.th Ed., Chapter 78, "Insulin, Glucagon And Diabetes Mellitus", (Prism Indian Edition), Prism-Saunders, Bangalore, Pp 980 -983). Diabetic Symptoms, which may arise due to pathological physiology of Insulin lack, are Polyuria, Polydipsia, Polyphagia, Asthenia and diminished utilization of carbohydrates for energy.
Complications of disease diabetes Mellitus involves many organs systems of the human body leading to many systemic complications like Diabetic Neuropathy, Diabetic Diarrhea, Urinary retention, Gustatory Swelling, Papillary Reflexes, Cardiac Autonomic Disturbances, Collagen Disturbances. Of these disorders 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.
In Ayurveda several herbal ingredients are mentioned for the treatment of diabetes (Madhumeha). Herbal ingredients such Gurmar leaves (Gymnema sylvestre), Methi seeds (Trigonella foenumgrecum), Vijayasar heartwood (Pterocarpus . marsupium), Jamun seeds (Eugenia jambolana), Karela (Momordica charantia) etc are few examples in this category. Various scientific investigations on these plants suggest their role in the care of diabetics (Atta-Ur-Rahman & Khurshidzaman, Journal Of Ethnopharmacology, 26 (1989); 1-55.). Many of the herbal formulations for diabetes available in the market are perhaps based on these leads. 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 Sy∑ygium 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 Myanmar and extends up to Afghanistan. It is generally cultivated as a roadside avenue tree as well.
In India, the decoction of kernels of Eugenia jambolana (EJ) are used as a household remedy for diabetes. These also form constituents of many herbal formulations for diabetes that are marketed in this country. The anti-hyperglycemic effect of aqueous and alcoholic extracts as well as lyophilized powder of these two plants was evaluated in diabetic animals using different doses of diabetogenic agents for varying duration (21- 120 days) so as to assess their effect in mild (plasma sugar>180 mg/dl, duration 21 days), moderate (plasma sugar>280 mg/dl, duration 120 days) and severe (plasma sugar>400 mg/dl, duration 60 days) diabetes mellitus (Grover.et.al.,Journal of Ethnopharmacology 2000; 73:461-470) One of the studies conducted by Rave elal., evaluated the hypoglycemic activity of different parts of Eugenia jambolana seeds such as whole seed, kernel, and seed coat on streptozotocin-induced rats. Administration of the ethanolic extract of kernel at a concentration of 100 mg/kg of body weight significantly decreased the levels of blood glucose, blood urea, and cholesterol, increased glucose tolerance and levels of total proteins and liver glycogen, and decreased the activities of glutamate oxaloacetate transaminase and glutamate pyruvate transaminase in experimental diabetic rats. Whole seed extracts exibhited a moderate hypoglycemic effect, and seed coat did not show any hypoglycemic effect. The hypoglycemic efficacy was compared with that of glibenclamide, a standard hypoglycemic drug (K. Rave et.al., Journal of Medicinal Food, 2004; 7(2):187-191)
The subject matter of the current invention describes extracts isolated from Eugenia jambolana 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.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.
DESCRIPTION OF THE FIGURES:
The present invention will become more fully understood from the detailed description given herein below, and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein;
Figure 1 : Shows the free radical scavenging ability of the Eugenia 70% ethanol extract comparable to ascorbic acid standard. Figure 2: Shows the percent polyphenol content of the Eugenia 70% ethanol extract comparable to gallic acid standard.
Figure 3: Shows the IC50 (μg/ml) value for a-glucosidase (0.2 EU) inhibition of the Eugenia 70% ethanol extract (60 μg/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 μg of Eugenia 70% ethanol 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 μg Eugenia 70% ethanol 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 μg of Eugenia 70% ethanol 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 μg Eugenia 70% ethanol 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 postprandial blood glucose level of diabetic Sprague dawley rats at different levels of Eugenia 70% ethanol extract intervention (50, 125 and 250 mpk) compared to that of baseline values over 5 weeks.
Figure 9: Shows the percent changes in fasting blood glucose level of diabetic Sprague dawley rats at different levels of Eugenia 70% ethanol extract intervention (50, 125 and 250 mpk) compared to that of baseline values over 5 weeks. Figure 10: Shows the percent changes in glycated hemoglobin level of diabetic Sprague dawley rats at different levels of Eugenia 70% ethanol extract intervention (50 and 125mpk) compared to that of baseline values over 5 weeks.
SUMMARY OF THE INVENTION:
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.
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 or organic solvent-based extraction of a specific therapeutically important phytochemical extract.
The therapeutic potential of the said extracts has been studied and confirmed through standard in vitro cell free and cell based assays.
It is a more specific aspect of the invention to provide a novel method of treating, preventing diseases or conditions characterized by increased bone resorption by administering a therapeutically effective amount of the composition containing the extract to a mammal in need of such therapy.
It is another object of the invention to provide a novel single medicinal extract or a combination of extracts thereof derived from Eugenia spp is preferably orally administrable but the invention contemplates topical, intradermal, intramuscular, parenteral or intravenous administrations thereof. These and other objects of the invention will be more fully understood and apparent from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto.
DETAILED DESCRIPTION OF THE INVENTION:
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.
In accordance with a further embodiment of the present invention, the potential plant is a member of the family Myrtaceae. hi another embodiment of the invention, 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 mat 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.
As used herein, "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. As used herein, "extract" refers to a concentrated preparation of the essential constituents of the medicinal plant. Typically, an extract is prepared by drying and powderizing the plant. 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.
In one embodiment of the invention, there is provided a process for obtaining a plant extract possessing hypoglycemic properties, the process 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, an ethanolic or an organic solvent, or a combination thereof, 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 70% ethanol extracts for glucose uptake and the in- vivo efficacy studies.
Extraction of the plant material by solvent extraction process:
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. If desired the plant material can be treated prior to extraction, for example, by drying, freezing, lyophilizing, or some combination thereof. If desired, the plant material can be fragmented and/or homogenized by some means such that a greater surface area is presented to the solvent. For example, 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 crashed or fragmented into smaller pieces.
The solvent used for the extraction process can be aqueous, alcoholic or organic, or a combination thereof. In one embodiment of the present invention, plant material is extracted with an aqueous solvent. Examples of suitable solvents include but are not limited to water, buffers, cell media, dilute acids or bases and the like. In an alternate embodiment of the invention, the plant material is extracted with an alcoholic solvent. Examples of suitable alcoholic solvents include, but are not limited to methanol, ethanol, n-propanol, iso-propanol, 2-butanol, ter-butanol, and combinations thereof. But preferably 70% ethanol water is used as solvent for extraction purposes. The coarse powder of herbal material is soaked in 70% ethanol 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.
Various extraction processes are known in the art and can be employed in the methods of the present invention. 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. Adequate contact of the solvent with the plant material can be encouraged by shaking the suspension. 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.
Also contemplated by the present invention are the modifications to the small-scale procedure that may be required during the scale up for the industrial level production of the extract. Such 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.
In yet another embodiment of the subject invention, concentration of the purified extracts or partially purified extracts by solvent removal from the original extract and/or fractionated extract, and/or purified extract. The techniques of solvent removal are known to those skilled in the art. and 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. Examples of various other organic solvents include but are not limited to, di-ethyl ether, hexane, heptane, dichloromethane, ethyl acetate, butyl alcohol, ether, acetone and the combinations thereof.
Tests to determine the free radical scavenging potential of the Eugenia extracts
Free radicals are constantly being generated in the body, as a result of the normal metabolic processes. Under physiological conditions, damage due to free radicals is countered by antioxidants. Sometimes, excessive free radical formation occurs in the body, and the antioxidant systems in the body cannot cope with the situations, i.e., the pro-oxidants overwhelm the antioxidants. This situation is known as oxidative stress. Thus, 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. The weight of experimental and human evidence supports a clear role for increased oxidant stress in many of the proposed biochemical pathways linked to the microvascular and microvascular complications of diabetes. In particular, very recent evidence has underscored the particular role the elevated glucose plays in oxidative modification of LDL by a superoxide dependent pathway, and has demonstrated that, in people with poorly controlled IDDM5 there is increased LDL oxidation associated with reduced antioxidant defenses.
There are several intriguing human studies, however, that show that administration of antioxidants stress plays a role in the reduced metabolic effects of insulin. In humans, the diabetogenic process appears to be caused by immune destruction of the beta cells; part of this process is apparently mediated by white cell production of active oxygen species. Diabetes can be produced in animals by the drugs alloxan and streptozotocin; the mechanism of action of these two drugs is different, but both result in the production of active oxygen species. Scavengers of oxygen radicals are effective in preventing diabetes in these animal models. Not only are oxygen radicals involved in the cause of diabetes, they also appear to play a role in some of the complications seen in long-term treatment of diabetes. Oberley et.al Free radicals and diabetes, Free Radic Biol Med. 1988; 5(2): 113-24. 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 mellitus, Nippon Ronen Igakkai Zasshi. 1990 Mar; 27(2): 149-54.
In the present invention 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 70% ethanol extract isolated from the seeds of the plant showed nearly 60% of free radical scavenging potency equivalent to that of ascorbic acid.
Estimation of total polyphenols and its hypoglycemic effects.
Several studies show that apple'polyphenols have a positive effect on diabetes and insulin resistance in animals and humans, hi 1987, European researchers announced that an apple-derived polyphenol, phlorizin, "completely normalized insulin response" in diabetic rats. In 2004, 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."
An Asian study in diabetic human volunteers showed that even weak polyphenols in apple juice produced "avoidance of sharp peaks" in blood glucose levels. Polyphenols from wine have favorable effects on glucose metabolism and insulin sensitivity, and many observational studies from around the world have shown that diabetes occurs less commonly among moderate drinkers than among abstainers. The polyphenolic polymers' have anti-oxidant effects, which provides synergistic benefits to persons with variousforms of diabetes.
In the current invention, the total polyphenol content of the Eugenia 70% ethanol 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 70% ethanols extract showed 53.4 ± 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.
Tests to confirm the Intestinal α-glucosidase inhibition potential
The inhibition of degradation of oligosacharrides (carbohydrates having 2 to 10 glucose residues connected by 1-4 or 1-6 α-D-glycosidic linkage) into monosaccharides by alpha- glucohydrolase-catalyzed enzymatic reactions was tested for Eugenia 70% ethanol 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 70% ethanol extract showed greater α-glucosidase inhibition potential (IC50 value of 32.79 μg/ml) relative to the commercially available α- glucosidase inhibitor, acarbose (IC50 value of 146.55μg/ml) for 0.2 α-glucosidase enzyme units at standard enzymatic reaction conditions.
In-vitro screening of Eugenia 70% ethanol extract for glucose untake
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 rnirnicking insulin, these drugs acts either by stimulating insulin release [Sulphonymrease], potentiating insulin action (thiazolidinedione). or lowering hepatic glucose production (biguanides). Various amounts of Eugenia 70% ethanol extract (0.034μg to 33.4μg) 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.
Use of the extract as a therapeutic composition:
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.
Thus, in one embodiment of the present invention, 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 inhibitor. A combination that demonstrates therapeutic index in comparison to the individual properties is considered to be an effective combination.
For compositions comprising two or more plant extracts, various ratios of the constituent plant extracts are contemplated. By a way of example, for a composition comprising two plant extracts, for example, extract A and extract B, the ratio of extract A to extract B can vary anywhere between 1:99 and 99:1. By "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. As would be apparent to one skilled in art, the effective amount may vary, depending upon, for example the disease to be treated and the form of administration. In general 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.
Modes of administration:
For administration to a mammal, 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 com starch, or alginic acid, binding agents, such as starch, gelatine or acacia, and lubricating agents, such as magnesium stearate, stearic acid or talc. The tablets can be uncoated, or they may be coated by known techniques in order to delay disintegration and absorption in. the gastrointestinal tract and thereby provide a sustained action over a longer period.
Various 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
Figure imgf000017_0001
Pharmaceutical or naturopathic formulations 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.
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.
In a * further aspect of the invention there is provided 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. The skilled addressee will appreciate that such 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. Thus 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. It is thought that a multi-functional role could be assigned to pharmaceutical formulations comprising two or more extracts possessing dissimilar therapeutic or prophylactic properties designed either for prophylaxis or for the treatment of more than one disease(s) in a mammal, particularly in a human.
The type of foodstuff or comestible to which at least an extract of the invention may be added includes any processed food such as confectioneries, 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, daily 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.
The invention will now be exemplified with reference to the following Examples section. It is to be understood that the examples are not to be construed as limiting the scope of the invention in any way.
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:
Example I
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 lOOμm. The bioactive extract is prepared from the seeds.70% ethanol 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 250C 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. Later 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.
Example 2: Free radical scavenging potential
Eugenia 70% ethanol extract isolated by the above mentioned method is tested 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 70% ethanol extract showed 85.2 ± 2.5 % free radical scavenging potency equivalent to ascorbic acid upon comparison, which is depicted in figure 1.
Example 3: Total polyphenols
The total polyphenol content of the Eugenia 70% ethanol 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 70% ethanols extract showed 53.4 + 2.25 % total phenol content equivalent to gallic acid, which is shown in figure 2.
Example 4: Intestinal α-glucosidase inhibition potential
The inhibition of degradation of oligosacharrides (carbohydrates having 2 to 10 glucose residues connected by 1-4 or 1-6 alpha-D-glycosidic linkage) into monosaccharides by alpha-glucohydrolase-catalyzed enzymatic reactions is tested for Eugenia 70% ethanol 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 70% ethanol extract showed greater α-glucosidase inhibition potential (IC50 value of 32.79 μg/ml) relative to the commercially available - α glucosidase inhibitor, acarbose (IC50 value of 146.55μg/ml) for 0.2 α-glucosidase enzyme units at standard enzymatic reaction conditions.
Example 5: Ih- vitro screening of Eugenia 70% ethanol 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 drags acts either by stimulating insulin release [Sulphonylurease], potentiating insulin action (thiazolidinedione) or lowering hepatic glucose production (biguanides). Various amounts of Eugenia 70% ethanol extract (0.034μg to 33.4μg) 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 0.334μg. 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 4- 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 (300μg/well, 30μg/well, 3μg/well and 0.3μg/well) in triplicates for 18 hours at 370 C and 5%CO2 lOOμl 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. For standard insulin response incubate cells with 5nM, 10 nM, 25 nM, 50 nM and 10OnM in KRH buffer in triplicate. To one set of wells to be treated with extract +insulin, 5nM of insulin in KRH buffer is added and in other set of well treated with extract alone, lOOμl of KRH buffer is added for 15 minutes at 37° C and 5%CO2. Glucose uptake reaction is initiated by adding 0.1 mM 2-deoxy glucose containing 2-deoxy [3H] glucose (final concentration 12.2 kBq/ml) and incubated for 1 hour at 370C and 5%CO2.. assay is terminated by adding 40 μM Cytochalasin B. The cells are washed three times with ice-cold KRH buffer (lOOμl). KRH buffer is removed and 20 μl 1% Triton X is added to each well to lyse the cells and incubate for 10 min at 37°C and 5%CO2. 200μl 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 70% ethanol extract is depicted in figure 4 to figure 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.
Example 6: In- vivo efficacy screening using Sprague dawley rats In-vivo efficacy screening of Eugenia 70% ethanol extract was done using Sprague dawley rats and the procedure undertaken is as follows:
Sprague dawley rats weighing ~250g with a variation of ± 20% of the mean weight are selected for In-vivo efficacy screening of Eugenia 70% ethanol extract. The number of animals per dose group is five and is kept for examination and acclimatization for a week period at the start of the study. The total number of dose groups is six along with vehicle control. The different groups are Gl -Vehicle control, G2-Diabetic control, G-3 Diabetic animals treated with Pioglitazone (Standard anti-diabetic drug). G-4 Eugenia 70% ethanol extract 50 mg/kg-body weight, G-5 Eugenia 70% ethanol extract 125 mg/kg-body weight, G-6 Eugenia 70% ethanol extract 200 mg/kg-body weight. Blood is collection under CO2 anesthesia by retro orbital (ROP) plexus method and FBG analysis. Rats are fed with 5 % glucose water for two days before STZ injection and are fasted for 12 hours before adrninistering streptozotocin. The rats are injected intra-peritoneal with 45mg/kg- body weight of streptozotocin in a citrate buffer ph: 4.5 and allowed for week recovery. Rats are fasted for 12 hours after 7th day of STZ injection and blood collected in heparin as the anti-coagulant, under CO2 anesthesia and analyzed for FBG. Rats that have the range of FBG beyond 250 mg/dl and above are considered for the study. On 8th day onwards, the in-vivo efficacy screening of Eugenia 70% ethanol extract at different concentrations is started and the dosing is 50, 125 and 200mg per kg body with 0.5 percent of carboxyl-methyl-cellulose (CMC) as vehicle.
The postprandial blood glucose lowering potential of Eugenia 70% ethanol extracts at different concentrations are illustrated in figure 8. 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 70% ethanol 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
Figure imgf000024_0001
Table 2. Post prandial blood glucose level Results.
The fasting blood glucose lowering potential of the Eugenia 70% ethanol extracts at different concentrations are illustrated in figure 9. The diabetic control rat group showed 246% increase in their fasting blood glucose levels compared to the baseline values of the same group at the start of the study. The group treated with pioglitazone showed 31.37% increase, while Eugenia 70% ethanol extract at 50, 125 and 200mpk showed 50% increase, 17.6% decrease and 39.36% increase respectively from their baseline values at the end of 35 days. The glucose lowering response of 125 and 200 mpk Eugenia 70% ethanol extracts are on par with the response of pioglitazone at 20mpk. The results have been represented in Table No. 2
Figure imgf000025_0001
Table 2: Fasting Blood Glucose level results
The glycated hemoglobin lowering potential of the Eugenia 70% ethanol extracts at different concentrations are illustrated in figure 10. Diabetic control rats showed 84.35% of HbAIc compared to the control rats (38.19%), pioglitazone treated rats (73.63%) 50mρk (50.04%) and 125mpk Eugenia 70% ethanol extract treated rats (86.30%).

Claims

5. Claims:
1. A method of treating or preventing diabetes by administering to a mammal a therapeutically effective non-toxic amount of an extract derived from the plant Eugenia characterized of hypoglycemic activity.
2. A method of obtaining a plant extract capable of delaying the onset and / or management of diabetes and related conditions thereof comprising the following steps: a. Obtaining plant material from one or more parts of me plant Eugenia preferably the seed. b. Obtaining an extract from the plant material by contacting the plant material with an aqueous, an ethanolic or an organic solvent, or a combination thereof, thereby providing one or more plant extracts possessing hypoglycemic activity. c. Analyzing the plant extracts for free radical scavenging potential, total polyphenols content, α-glucosidase inhibition potential and for insulin mimetic and sensitization activity. d. Selecting plant extracts having one or both of these activities.
3. A method according to claim 1 and 2, wherein the plant extracts are derived from the plant Eugenia jambolana.
4. A method according to claim any of the preceding claims wherein, the plant extracts are extracted from leaves, seeds, roots, stems, flowers, or various combinations thereof but preferably the seed of the plant.
5. A method according to claim 1 and 2, wherein the extract possessing hypoglycemic activity is derived by contacting the plant material with a solvent for a time period sufficient to ensure adequate exposure of the solid plant material to the solvent such that the potential extract is taken up by the solvent.
6. A method according to the preceding claim, wherein the solvent is selected from ethanol, methanol, 1-propanol, 2-propanol, ethyl acetate, and the preferred mixture containing 30 parts and 70parts by volume of water and ethanol respectively.
7. A method according to claim 1 and 2, wherein the bioactive extract comprises at least 80 % free radical scavenging potential equivalent to ascorbic acid.
8. A method according to claim 1 and 2, wherein the bioactive extract comprises at least 50% of total polyphenols content equivalent to that of gallic acid.
9. The successful plant extracts derived from the extraction process demonstrating a) insulin mimetic activity b) insulin sensitizing activity c) alpha glucqsidase activity in combination with a pharmaceutically acceptable carrier.
10. Use of the Eugenia plant extract according to at least one of the claims 1 to 9 as a medicament.
11. Use of the Eugenia plant extract according to at least one of the claims 1 to 9 as an antioxidant.
12. Use of the Eugenia plant extract according to at least one of the claims 1 to 9 for the production of a medicament with hypoglycemic activity.
13. A composition comprising one or more plant extracts that are capable of delaying the onset and/or management of diabetes derived from the plant belonging to the plant sp. Eugenia.
14. A composition comprising one or more plant extracts, wherein the administration of the therapeutic composition can be formulated as pharmaceutical or naturopathic formulation such as phytoceuticals or nutraceuticals, for oral, topical, rectal, parenteral administration or for administration by inhalation or spray.
15. A composition comprising one or more plant extracts, wherein 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.
16. A composition comprising one or more plant extracts, wherein the extracts can be formulated as phytoceuticals or nutraceuticals not limiting to teas, tonics, juices, syrups and nutraceuticals providing nutritional benefits in the form of foods, beverages, supplements and the like.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR6114M (en) * 1967-01-19 1968-06-17
FR2465484A1 (en) * 1979-09-21 1981-03-27 Urwerg Suzanne Extract of e.g. eugenia jambolana lamarck - has antidiabetic, hypoglycaemic, hypolipaemic, weight reducing anorexic and anti-cataract activity
WO2000074698A1 (en) * 1999-06-04 2000-12-14 Indian Council Of Medical Research Eugenia jambolina fruit extracts for treating diabetes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR6114M (en) * 1967-01-19 1968-06-17
FR2465484A1 (en) * 1979-09-21 1981-03-27 Urwerg Suzanne Extract of e.g. eugenia jambolana lamarck - has antidiabetic, hypoglycaemic, hypolipaemic, weight reducing anorexic and anti-cataract activity
WO2000074698A1 (en) * 1999-06-04 2000-12-14 Indian Council Of Medical Research Eugenia jambolina fruit extracts for treating diabetes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ARAI I ET AL: "Improving effects of the extracts from Eugenia uniflora on hyperglycemia and hypertriglyceridemia in mice.", JOURNAL OF ETHNOPHARMACOLOGY 15 DEC 1999, vol. 68, no. 1-3, 15 December 1999 (1999-12-15), pages 307 - 314, XP002434035, ISSN: 0378-8741 *
KOHLI K R ET AL: "EUGENIA-JAMBOLANA A PLANT DRUG WITH POTENTIAL ANTIDIABETIC PROPERTY A REVIEW", JOURNAL OF SCIENTIFIC RESEARCH IN PLANTS AND MEDICINES, YOGI PHARMACY, HARDWAR,, IN, vol. 6, no. 1-4, 1985, pages 21 - 28, XP000940820, ISSN: 0253-7249 *
SAGRAWAT, H. ET AL: "Pharmacological potential of Eugenia jambolana: a review", PHARMACOGNOSY MAGAZINE , 2(6), 96-105 CODEN: PMHACG; ISSN: 0973-1296, 1 June 2006 (2006-06-01), XP009083973 *
TEIXEIRA, C. C. ET AL: "Absence of antihyperglycemic effect of jambolan in experimental and clinical models", JOURNAL OF ETHNOPHARMACOLOGY , 71(1,2), 343-347 CODEN: JOETD7; ISSN: 0378-8741, 2000, XP002434036 *

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