WO2008004119A2 - Extraits d'eugenia jambolana pour le traitement de l'ostéoporose, et procédé d'extraction correspondant - Google Patents

Extraits d'eugenia jambolana pour le traitement de l'ostéoporose, et procédé d'extraction correspondant Download PDF

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WO2008004119A2
WO2008004119A2 PCT/IB2007/002064 IB2007002064W WO2008004119A2 WO 2008004119 A2 WO2008004119 A2 WO 2008004119A2 IB 2007002064 W IB2007002064 W IB 2007002064W WO 2008004119 A2 WO2008004119 A2 WO 2008004119A2
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agt006
extract
extracts
drso
bone
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PCT/IB2007/002064
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WO2008004119A3 (fr
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Villoo Morawala Patell
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Avestha, Gengraine, Technologies, Pvt., Ltd.
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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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention relates to compositions and methods for preventing, treating, or managing osteoporosis or other related disorders such as bone loss, bone fracture, glucocorticoid induced osteoporosis, Pagets disease, osteoarthritis, peri-prosthetic osteolysis, cartilage degeneration, osteogenesis imperfecta and the like, comprising administration of a prophylactically and therapeutically effective amount of Eugenia jambolana plant or extracts thereof to a mammal in need of such therapy.
  • the mammal is human and the compositions comprise of single extract or a combination of extracts thereof.
  • the present invention further relates to extracts which are isolated from different parts of Eugenia jambolana plant, the preparation of such extracts, the medicaments containing said extracts, and the use of these extracts and constituents for the preparation of a medicament.
  • the present invention also relates to the process for preparing the extracts from the seeds of Eugenia jambolana plant.
  • Osteoporosis is a skeletal disorder that is characterized by low bone mass and micro- architectural deterioration of bone tissue. Affecting 200 million individuals world-wide, osteoporosis is the most common metabolic bone disorder which leads to an increased level of bone fragility and susceptibility to fracture (Walker-bone, et al. 2002; Lin and Lane, 2004). A. Classification of Osteoporosis Based on its etiology, osteoporosis is categorized as a primary or secondary disease.
  • osteoporosis involves the onset of osteoporosis as a result of an existing condition such as an endocrine disorder, the use of certain medications, a hematopoietic disorder, immobilization, or a nutritional, gastrointestinal or connective tissue disorder (Lin and Lane, 2004).
  • Primary osteoporosis is further subdivided into two types. Type I generally occurs in postmenopausal women and is attributed to loss of gonadal hormone function, such as estrogen deficiency associated with menopause (Lin and Lane, 2004; Simon, 2004).
  • Type II osteoporosis generally called as senile osteoporosis is age-related, affecting both men and women over the age of 60.
  • Bone and Lane 2004 Through the assessment of bone mineral density (BMD; in g/cm 2) using dual energy x-ray absorptiometry (DEXA), the World Health Organization has defined osteoporosis as a BMD more than 2.5 standard deviations below the mean of normal, healthy individuals at their peak bone mass (Lin and Lane, 2004; Simon, 2004; Christodoulou and Cooper, 2003).
  • BMD bone mineral density
  • DEXA dual energy x-ray absorptiometry
  • Bone remodeling occurs at specific sites on the bone surface known as basic multicellular units and is carried out by osteoclasts (bone resorption cells) and osteoblasts (bone formation cells) (Tolstoi 2004, Manolagas and Weinstein, 1999).
  • Both osteoblasts and osteoclasts are derived from precursors originating in the bone marrow and the formation and activation of these cells is regulated by cytokines and growth factors also produced in the bone marrow, which are in turn controlled by systemsic factors and mechanical stimuli (Manolagas and Weinstein 1999).
  • the bone remodeling cycle is highly dependent on a delicate balance between regulatory signaling and cellular activity. Loss of the capacity to recruit active osteoblasts or deactivate osteoclasts results in a net bone loss and can lead to the onset of osteoporosis. ///.
  • osteoporosis Although numerous risk factors have been identified to increase the likelihood of developing this disease, including Caucasian race, advanced age, female gender, history of fracture, smoking and alcoholism, the exact cause of osteoporosis has not yet been identified. Despite this, numerous theories have been proposed in an attempt to explain its etiology. Some theories regarding the etiology of osteoarthritis include bone cell senescence, lifestyle factors (primarily exercise and nutrition) and loss of vitamin D metabolism with age (Tsai, et al., 1984). The latter hypothesis infers that aging leads to an impaired metabolism of vitamin D. Activated vitamin D is a signaling molecule that is largely involved in the regulation of intestinal calcium absorption Tsai, et al., 1984).
  • antiresorptive drugs which slow the progressive thinning of bone
  • bone forming drugs which help to rebuild the skeleton
  • drugs with a more complex mechanism of action drugs used to treat osteoporosis.
  • calcium and vitamin D supplements might also be prescribed to ensure adequate intake and to ensure maximum effectiveness of the drug therapy.
  • Bisphosphonates inhibit bone resorption. They are currently the first choice of treatment in a variety of bone metabolism disorders characterized by high bone resorption.
  • SERM Selective estrogen receptor modulators
  • a new drug has been approved for the treatment of osteoporosis - this time one that improves bone formation, as opposed to the action of available drugs that is usually the prevention or slowing bone resorption. It is teriparatide, a 34-amino-acid polypeptide produced by a recombinant DNA technique, which represents the biologically active part of human parathyroid hormone. It has to be given once daily by subcutaneous injection. They treat osteoporosis by stimulating bone-forming cells called osteoblasts. It has a dramatic effect on bone, increasing bone mineral density in the spine by as much as 13% in 18 months and reducing the risk of fracture by as much as 90%. The reason that patients are not using this drug is "cost". Forteo costs about $600 a month, and it also must be injected every day. For those reasons, it's generally only prescribed for patients with severe osteoporosis, or who have already had one or more fractures.
  • Seeds of Eugenia contain fatty oils (30g/kg) including lauric acid (2.8%), myristic acid (31.7%), palmitic acid (4.7%), stearic acid (6.5%), oleic acid (32.2%), linoleic acid (16.1%), malvalic acid (1.2%), sterculic acid (1.8%) and vernolic acid (3%) and phytosterols such as ⁇ - Sitosterol.
  • Others constituents are tannins (6%) predominantly corilagin, ellagitannins, ellagic acid, galloyl- galactoside and gallic acid. Additionally phenolic compounds like ferulic acid, quercetin, veratrole, guajacol, and caffeic acid have been identified as constituents of Eugenia jambolana.
  • Jamun is believed to be of special use in the treatment of diabetes.
  • the extracts of the bark, seeds and also leaves are used.
  • Aqueous extract of the seeds is reported to cause marked, prolonged decrease in blood sugar, when injected into dogs.
  • Experiments carried out at Central Drug Research Institute, Lucknow, showed that oral administration of dried alcoholic extract of the seeds appeared to diabetic patients reduces the level of blood sugar and glycosuria. Fresh seeds appeared to be superior to dried ones
  • JSEt Jamun seed extract
  • GSH reduced glutathione
  • SOD superoxide dismutase
  • CAT catalase
  • the invention relates to compositions and methods for preventing, treating, or managing osteoporosis other related disorders such as bone loss, bone fracture, glucocorticoid induced osteoporosis, Pagets disease, osteoarthritis, peri-prosthetic osteolysis, cartilage degeneration, osteogenesis imperfecta and the like, comprising administration of a prophylactically and therapeutically effective amount of Eugenia jambolana plant or extracts thereof to a mammal in need of such therapy.
  • the mammal is human and the compositions comprise of single extract or a combination of extracts thereof.
  • the present invention further relates to extracts which are isolated from seeds of Eugenia jambolana plant, the preparation of such extracts, the medicaments containing said extracts, and the use of these extracts and constituents for the preparation of a medicament.
  • the present invention also relates to the process for preparing the extracts from Eugenia jambolana plant seeds.
  • the process comprises of: • Cleaning the Eugenia jambolana plant material.
  • compositions/medicaments may contain a pharmaceutically acceptable carrier, excipient, or diluent.
  • the compositions can be included as unit dosage suitable for parenteral, oral, or intravenous administration to a human.
  • the compositions are dietary supplements, food compositions or beverage compositions suitable for human or animal consumption.
  • the invention further describes the biotherapeutic potential of various extracts of Eugenia jambolana as described above, by studying their performance in cell based assay models.
  • Fig 1 The cell viability for the extract AG07, AG08, AG09, AGIO, AGl 1 and AG12
  • Fig 2 The CTX release for plant extract AG07-AG12 for the resorption experiment
  • Fig 3 The cell viability for plant extract AG07-AG12 for the resorption experiment
  • Fig 4 The CTX release for plant extract AG09 lead characterization
  • Fig 5 The cell viability for plant extract AG09 lead characterization Detailed description of the Invention
  • a method for treating a disease in a mammal which comprises administering to the said mammal an effective non-toxic amount of at least an extract from Eugenia jambolana as defined herein.
  • the mammal is a human being.
  • treating a disease means treating, that is to say, alleviating symptoms of the disease and may also mean managing a disease in the sense of preventing such a disease state either advancing ie getting worse or becoming more invasive, or slowing down the rate of advance of a disease.
  • the mammal is human and the said extract comprises a single extract from a plant part of Eugenia jambolana or a combination of extracts therefrom as detailed herein.
  • the present invention further relates to extracts, which may be isolated from seeds of the Eugenia jambolana plant, the preparation of such extracts, medicaments comprising such extracts, and the use of these extracts and constituents for the preparation of a medicament.
  • Extracts of the present invention can be isolated from seeds of Eugenia jambolana, using conventional organic solvent extraction and supercritical fluid extraction technology.
  • extracts of the invention capable of functioning in a prophylactic or therapeutic manner as outlined herein can be extracted from any Eugenia jambolana plant, depending on the end purpose that is required of the extract.
  • a process for preparing extracts of the invention from plant parts of Eugenia jambolana that comprises: • Pulverizing selected plant material to a powder;
  • the choice of selected plant material may be of any type but is preferably the seeds of the Eugenia jambolana plant.
  • the solvent extraction process may be selected from direct 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). Typically, the extraction process is as outlined herein.
  • mouse osteoblastic cells MC3T3 (mouse clavarial osteoblast like cells) were seeded at a density of 20,000 cells in 96 well plates and cultured for one day prior to the addition of the plant extracts.
  • the stock solutions of the extracts dissolved in DMSO were diluted to 3 different concentrations.
  • the cells after addition of the extracts are cultured for 3 days and cell viability is measured by a colorimeti ⁇ c based cell viability assay as exemplified in the detailed description.
  • separate cytotoxicity tests, stability tests and the like can be conducted to evaluate the toxicity of the extracts or compositions can be conducted.
  • the therapeutic compositions of the invention will need to meet certain criteria in order to be suitable for human or animal use and to meet the regulatory requirements.
  • standard in-vivo and in vitro tests can be conducted to determine the information about the metabolism and pharmacokinetics of the compositions, including data on the drug- drug interactions where appropriate, which can be used to design human clinical trials.
  • the present invention further contemplates that where toxicity is a factor, for example, in patients who cannot tolerate optimal or standard therapeutic dosages, or in cases where the patient's metabolism is compromised sub-optimal doses would be preferred.
  • osteoclasts are largely derived from CD 14 positive monocytes (J.Haemotol, 1999 JuI; 106(l):167-70). Clonal analysis of haematopoietic cells by surface phenotypes has been used to further identify osteoclast precursor by characterizing osteoclast like cells distinct from other haematopoietic progenitors (Lee et.al; 1992b Muguruma & Lee; 1988). Surface phenotype analysis has shown that human osteoclasts are derived from CD 14 monocytes (Massey & Flanagan, 1999). CD 14 marker is strongly expressed on monocytes, the putative osteoclast precursor in peripheral blood and CD 14 positive monocytes have been selected for osteoclastogenesis. (Clin.Sci (Lond) 2000, Aug, 99(2): 133-40).
  • a method of screening of plant extracts suitable for inclusion in the therapeutic compositions comprising (a) providing one or more plant extract isolated with a specific solvent (b) analyzing the one or more extract for their inhibitory activity on bone resorption (c) selecting the extracts that decrease the activity of bone resorption by atleast 40%, as plant extracts suitable for inclusion in the therapeutic compositions.
  • Bone particle-based assays Bone particles, obtained by grinding of 45Ca- or [3H] proline-labeled bones, are cultured with osteoclasts. 45Ca or [3H] release is measured at the end of the cultures (Oreffo et al. 1988). Bone slice assay. This widely used assay is based on the observation that isolated osteoclasts make resorption pits on slices of devitalised dentine or bone (Boyde et al.1984) and that these pits resemble Howship's lacunae.
  • Various formats may be used if the potential extracts are to be tested against a specific set of cultured osteoclast cells.
  • the assays may be adapted in order to facilitate the simultaneous testing of many potential extracts. Such techniques are being constantly developed and the use of such techniques to identify the potential extract activity is considered to be within the scope of the present invention.
  • osteoblast culture systems that have been developed include cultures containing osteoblast or osteoblast-like cells from different species, bones of different ages, and a variety of anatomical sites and pathological states. Systems have also been developed for specific cell populations, such as osteoprogenitor cells and osteocytes. Several recent articles have also discussed various osteoblast cell culture models and provide some critical commentaries about their use (Marie, 1994; Rodan et al. 1994; Gundle & Beresford, 1995, Parfitt,1995; Roby, 1995). In the present invention MC3T3 cell line was used. These cells are murine osteogenic mesenchymal precursor cells, which can be differentiated into osteoblasts by ascorbic acid and beta-gylcerol phosphate.
  • a plant extract that increases activity of the osteoblast cells comprising (a) providing one or more plant extract isolated with a specific solvent (b) analyzing the one or more extract for their activity on bone formation (c) selecting the extracts that promote the activity of bone formation.
  • a colorimetric assay with o-cresolphthalein is commonly used to measure calcification in TCA extracts of cultured bones (Gronowicz et al. 1989).
  • Calcein a fluorescent dye that stains calcium phosphate deposits (Hock et al. 1968), can be used to measure calcification in mineralising cell cultures. Calcification, however, can be increased by bone damage or death (Ramp & Neuman, 1971). Therefore, the bone should also be checked by histological examination. Several histological methods can be used to assess bone morphology (Malluche & Faugere, 1986).
  • alkaline phosphatase which is simple to measure biochemically.
  • Its expression pattern in osteoblasts Doty & Schofield, 1976; Stein et al. 1996) and its involvement in mineralisation have been extensively documented (Wuthier & Register, 1984).
  • many cell types in bone or marrow stroma contain alkaline phosphatase, such as hypertrophic chondrocytes (Wuthier & Register, 1984) and adipocytes (Beresford et al. 1993), while fibroblastic cells also have a low concentration of this enzyme.
  • the alkaline phosphatase activity can provide a good indicator of osteoblast cells if the cartilage and marrow are removed by dissection.
  • the present invention evaluates the bone formation that has occurred through Alkaline phosphatase assay and the proliferation that has occurred through Alamar blue assay.
  • Suitable cell lines such as ST2 (mature monocytes and macrophages capable of differentiating into osteoclasts ,MLC-6 (osteoclast like cell line derived from mouse, MC3T3-E1 (mouse calvaria, Sudo et al 1983), MBA- 15 (Clonal marrow stromal cell line ) and the like.
  • Osteoblast cell lines include 2T3 (osteoblast cell line), AHTO, HOBIT cell lines and the like can be used for the cell based assays. These cell lines can be obtained from ATCC or various other commercial sources. The invention premeditates the use of such suitable osteoclast and osteoblast cell lines for conducting the cell based assays.
  • compositions suitable for use in the treatment of Osteoporosis comprising at least one extract as isolated from a Eugenia jambolana, in admixture with a pharmaceutically acceptable carrier.
  • the at least one extract is selected from those listed in Table 1.
  • compositions may comprise of two or more plant extracts of the invention in any concentration, which is capable of giving rise to a therapeutic effect.
  • therapeutic compositions can comprise plant extracts of Eugenia substantially devoid of undesirable contaminating compounds.
  • the plant extracts may have, for example, undergone a number of solvent extraction steps substantially to separate out undesirable components from desirable components such as those alluded to in the examples and aforementioned tables.
  • the invention thus further provides a method for the treatment of Osteoporosis or conditions, which are characterized by increased bone resorption in mammals, including humans, which comprises the use of a clinically useful amount of an extract selected from those listed in Table 1 , in a pharmaceutically useful form, once or several times a day or in any other appropriate schedule for example, orally, or intravenously or by delivery to the lungs in a dry or "wet" spray.
  • the amount of compound of extract required to be effective in the treatment Osteoporosis or conditions, which are characterized by increased bone resorption will, of course, vary with the disease being treated and is ultimately at the discretion of the medical or veterinary practitioner.
  • the factors to be considered include the condition being treated, the route of administration, and nature of the formulation, the mammal's body weight, surface area > age and general condition and the particular compound to be administered.
  • a suitable effective dose of an extract of the invention generally lies in the range of about 500 mg twice daily. Whilst it is possible for the active extract to be administered alone, it is preferred to present the active extract in a pharmaceutical formulation.
  • Formulations of the present invention, for medical use comprise an extract of the invention together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients.
  • the carrier(s) should be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and substantially non-deleterious to the recipient thereof.
  • the present invention therefore, further provides a pharmaceutical formulation comprising at least one extract selected from those listed in table 1, together with a pharmaceutically acceptable carrier therefore.
  • any pharmaceutical formulation comprising an active extract of the invention can include at least one active extract purified from an extract derived from a Eugenia species.
  • a pharmaceutical formulation may contain more than one active extract derived from two or more Eugenia species.
  • Formulations according to the present invention include those suitable for oral or intravenous administration.
  • Intravenous formulations including at least one extract of the invention and may also be administered in the form of suitable liposomal or niosomal preparations or other suitable delivery vehicle.
  • Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glycerol mono-stearate and sodium laury sulphate.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active extracts(s) into association with a carrier, which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active extract(s) into association with a liquid carrier or a finely divided solid carrier or both and then, if necessary, shaping the product into desired formulations.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets, tablets, lozenges, comprising the active ingredient in a flavoured based, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
  • Each formulation generally contains a predetermined amount of the active extract; as a powder or granules; or a solution or suspension in an aqueous or nonaqueous liquid such as syrup, an elixir, an emulsion or draught and the like.
  • a tablet may be made by compression or moulding optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing an a suitable machine the active extract in a free-flowing form such as a powder or granules, optionally mixed with a binder, (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycolate, cross- linked povidone, cross-linked sodium carboxymethyl cellulose), surface active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered extract moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile.
  • a syrup may be made by adding the active extract to a concentrated, aqueous solution of a sugar; for example sucrose, to which may also be added any necessary ingredients.
  • a sugar for example sucrose
  • Such accessory ingredient(s) may include flavourings, an agent to retard crystallisation of the sugar or an agent to increase the solubility of any other ingredients, such as a polyhydric alcohol for example glycerol or sorbitol.
  • the formulations of this invention may further include on or more accessory ingredients) selected from diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including antioxidants) and the like.
  • compositions are dietary supplements, food compositions or beverage compositions suitable for human or animal consumption.
  • a comestible that is to say, a foodstuff comprising at least an extract of the invention, typically in dried form, such as in a lyophilized form selected from those listed in Table 1 herein.
  • comestibles 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 muesli bars, compressed dried seed bars, biscuits, dairy products such as yoghurts, milk and milk-based products such as custards, cream, cheese, butter and creme fraiche, simulated dairy food products such as margarine, olive oil-based spreads, and low fat cream substitutes such as Elmlea products, seed 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, vegetable, seed and nut oils, seeded oils such as sunflower oil, rapeseed oil, olive oil, walnut, hazelnut, and sesame seed oil and the like, and frozen confections such as ice creams,
  • a suitable effective dose of an extract of the invention to be included in a comestible generally lies in the range 500 mg twice daily.
  • the vapors of the solvent from the flask passed through the inlet of the extractor and condensed.
  • the condensed (distilled) solvent was collected in the Extractor (body) thus extracting the compounds from it.
  • the extractor was completely filled with solvent, it got drained in the flask. This process is continuous as long as there is stable heat and water circulation.
  • the extraction was continued for 4 - 6 hours, 4-5 cycles per hour. After four to six hours the mantle was switched off and the water flow was stopped.
  • the extract was concentrated by fitting the flask containing the extract with the empty soxhlet extractor body that in turn was fitted tightly with the water-cooled condenser. Continuous water flow was maintained and the flask was heated till the solvent from the flask was distilled and collected in the extractor body up to a level. (One inch below the inlet). The temperature was reduced to avoid charring as the volume of the solvent reduced in the flask. The distilled solvent collected in the extractor was transferred to the solvent bottles and label appropriately. The process was continued till only very little solvent was left in the flask and no charring had occurred. The extract in the flask were swirled and were dried under vacuum followed by lyophilizing the obtained extracts between 50 to 500 m Torr (optimum HO m Torr) and between -30 to -80° C temperature (optimum -60° C).
  • Direct reflux extraction method 10 - 300 grams of powdered plant material was weighed into the round-bottomed extractor flask. 50 - 1000 ml of solvent was added in to the flask and placed on the mantle along with few (3-4) ceramic chips. The flask is fitted with a water-cooled condenser. The mantle was switched on and the desired temperature was set. During extraction the solvent boils in presence of plant material leading to the extraction of compounds from the plant material. This process is continuous as long as there is stable heat and water circulation. The extraction was continued for 4 - 6 hours, A- 5 cycles per hour. After four to six hours the mantle was switched off and the water flow was stopped. After cooling the plant material was removed by filtration through a cotton plug.
  • the extract was concentrated by fitting the flask containing the extract with the empty soxhlet extractor body that in turn was fitted tightly with the water-cooled condenser. Continuous water flow was maintained and the flask was heated till the solvent from the flask was distilled and collected in the extractor body up to a level. (One inch below the inlet). The temperature was reduced to avoid charring as the volume of the solvent reduced in the flask. The distilled solvent collected in the extractor was transferred to the solvent bottles and label appropriately. The process was continued till only very little solvent was left in the flask and no charring had occurred. The extract in the flask were swirled and were dried under vacuum followed by lyophilizing the obtained extracts between 50 to 500 m Torr (optimum HO m Torr) and between —30 to -80° C temperature (optimum -60° C).
  • Extract ID contains the first six letters of the plant code followed by the part of the plant used. This is followed by type of extraction, extraction temperature, solvent and the percentage of the solvent used. Which is followed by the date, month and year of extraction for the identification of the three extracts, which are the subject matter of the invention.
  • Example l ' 2OO grams of the plant material was extracted with 1000 ml of solvent at 65°
  • Table 1 Representative extracts of the seeds of Eugenia jambolana with different solvents
  • Example 2 100 grams of the solvent was extracted with 500 ml of solvent at 85° C followed by lyophilizing the obtained extracts between 50 to 500 m Torr (optimum 110 m Torr) and between -30 to -80° C temperature (optimum -60°-C)
  • Table 2 Representative extracts of the seeds of Eugenia jambolana with different solvents
  • Example 3 20 g of the plant extracted with 100 ml of the solvent at 85° C followed by lyophilizing the obtained extracts between 50 to 500 m Torr (optimum 110 m Torr) and between -30 to -80° C temperature (optimum -60° C).
  • Table 3 Representative extracts of the seeds of Eugenia jambolana with different solvents
  • the plant extracts were tested in three concentrations to examine the toxicity of the extracts to MC3T3 cells.
  • the stock solutions of the extract were diluted 1 :100, 1:1000 and 1:10000 in the medium for the cells.
  • the concentrations have been chosen so that the concentration of DMSO used for making the stock solutions is not higher than 1%.
  • MC3T3 cells were seeded at a density of 20000 cells per well in 96 well plate. The cells were cultured for one day before the plant extracts were added in the three dilutions. The cells were after the addition of the extracts cultured for three days. At the end of the culture period the cell viability was measured by the AlamarBlue assay.
  • AN09 and AN 15 is not toxic any of the dilutions, while AN21 is a little toxic at both 1:100 and 1:1000.
  • the toxicity of extract AN21 is low and the dilution 1 :1000 and 1 :10,000 was used for the hit screening in the bone resorption assay and in the bone formation assay.
  • Collagen type I makes up >90% of the organic matrix of bone.
  • Collagen type I is composed of linked type I collagen molecules, -with the amino- and carboxy-terminal regions of a type I collagen molecule linked to the helical region of adjacent collagen molecules through pyridinium cross-links, deoxypyridinoline (Dpd) and pyridinoline (Pyd).
  • Dpd deoxypyridinoline
  • Pyd pyridinoline
  • telopeptides consist of short peptide sequences from the helical domain of a type I molecule and the telopeptide region of an adjacent molecule, joined by a pyridinium cross-link (either Pyd or Dpd).
  • telopeptides released from bone are excreted unchanged in urine, and the remainder are metabolized by the liver and kidneys.
  • Commercial assays have been developed for 3 forms of collagen type I telopeptide: NTX, CTX, and ICTP (a slight variant of CTX).
  • the resorption process can be studied in vitro by culturing bone cells on devitalized slices of bone or dentin.
  • the osteoclasts were isolated as CD 14 positive monocytes and differentiated into osteoclasts for 10-12 days. Hereafter, the osteoclasts were lifted by trypsin, counted and seeded on bovine bone slices at a density of 40000 cells per bone slice. The osteoclasts were seeded on the bone slices for one day before the plant extracts were added. The osteoclasts were hereby allowed to attach and initiate resorption.
  • the plant extracts were added in two concentrations 1:10,000 and 1:100,000 dilutions because the toxicity study in the MC3T3 cells had shown that these concentrations were not toxic to the cells.
  • Bafilomycin is a V-ATPase blocker and it inhibits the osteoclastic bone resorption approximately 80% compared to DMSO (the negative control will be set to 100 % on the chart.
  • the bone resorption experiment was ended after 5 days of incubation and the cell viability was measured by the use of the AlamarBlue assay. Bone resorption was evaluated as the determination of the CTX release.
  • Plant extract AN09 which is Eugenia jambolana extract made with hexane, is not toxic at either dilution. However, the extract has a small inhibitory effect on the bone resorption at dilution 1:1000. AN09 inhibits bone resorption ⁇ 30% compared to the control at 1 :1000. Plant extract ANl 5, which is Eugenia jambolana extract made with acetone, is a little toxic at both dilution ( ⁇ 20% compared to the control) and the extract has no effect on bone resorption. Plant extract AN21, which is Eugenia jambolana extract made with water, has no effect on bone resorption.
  • MC3T3 cell lines were used for the bone formation studies. These cells are murine osteogenic mesenchymal precursor cells, which can be differentiated into osteoclasts by ascorbic acid and beta-glycerol phosphate.
  • the osteoblasts (MC3T3 cells) were seeded at a density of 75000 cells per well in 24 plates. The cells were culture for one day before the plant extracts were added. Then the osteoblasts were cultured for 14 days in medium supplemented with ascorbic acid and beta-glycerol phosphate (AA and bG) and in the presence of the plant extracts (1 :10,000 and 1:100,000 dilution)
  • BMP-2 bone morphogenic protein -2
  • BMP-2 is used as a positive control, while the treatment without ascorbic avid and beta-glycerol phosphate (without AA and bG) is the negative control.
  • BMP-2 induces the MC3T3 precursor cells to differentiate into osteoblasts and the ALP activity is induced compared to the negative control.
  • plant extract AN21 (dilution 1 : 10,000) has a positive osteogenic effect on the MC3T3 cells.
  • the ALP activity is a little higher for AN21 compared to the positive control, BMP-2.
  • AN21 was not toxic to the cells at any of the dilutions.

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Abstract

L'invention concerne des compositions et des procédés de prévention, traitement ou prise en charge de l'ostéoporose ou d'états caractérisés par un accroissement de la résorption osseuse, par administration à un mammifère justifiant un tel traitement, d'une quantité prophylactiquement et thérapeutiquement efficace d'Eugenia jambolana ou d'extraits d'Eugenia jambolana. De préférence, le mammifère et un humain, et les compositions comprennent un extrait unique ou une combinaison d'extraits d'ail. L'invention concerne également des extraits isolés à partir d'Eugenia jambolana, l'élaboration de tels extraits, les médicaments contenant ces extraits, et l'utilisation de ces extraits et constituants pour l'élaboration d'un médicament. L'invention concerne enfin le procédé d'élaboration des extraits à partir de diverses parties de la plante.
PCT/IB2007/002064 2006-07-06 2007-07-09 Extraits d'eugenia jambolana pour le traitement de l'ostéoporose, et procédé d'extraction correspondant WO2008004119A2 (fr)

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IN1194/CHE/2006 2006-07-06
IN1194CH2006 2006-07-06

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WO2008004119A2 true WO2008004119A2 (fr) 2008-01-10
WO2008004119A3 WO2008004119A3 (fr) 2008-07-31

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103926108A (zh) * 2013-01-11 2014-07-16 陈军 硬组织切片取骨机

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR6114M (fr) * 1967-01-19 1968-06-17
DE2159923A1 (de) * 1971-12-02 1973-06-14 Schwabe Willmar Verfahren zur gewinnung von stoffen mit insulinaehnlicher wirkung aus pflanzen und diese stoffe enthaltende arzneipraeparate
FR2465484A1 (fr) * 1979-09-21 1981-03-27 Urwerg Suzanne Extrait vegetal a proprietes antidiabetiques et son procede de preparation
WO1997028813A1 (fr) * 1996-02-06 1997-08-14 Institut Malgache De Recherches Appliquees Melanges isolables a partir de graines d'eugenia jambolana lamarck, leur preparation et l'utilisation de ces melanges et certains de leurs constituants comme medicaments
WO2000074698A1 (fr) * 1999-06-04 2000-12-14 Indian Council Of Medical Research Produit therapeutique a base de plantes
KR20020084873A (ko) * 2001-05-04 2002-11-13 알앤엘생명과학주식회사 정향피 추출물을 유효성분으로 함유하는 호르몬 대체치료용 조성물
JP2005298450A (ja) * 2004-04-15 2005-10-27 Taisho Pharmaceut Co Ltd 更年期症候群の予防又は治療剤組成物

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR6114M (fr) * 1967-01-19 1968-06-17
DE2159923A1 (de) * 1971-12-02 1973-06-14 Schwabe Willmar Verfahren zur gewinnung von stoffen mit insulinaehnlicher wirkung aus pflanzen und diese stoffe enthaltende arzneipraeparate
FR2465484A1 (fr) * 1979-09-21 1981-03-27 Urwerg Suzanne Extrait vegetal a proprietes antidiabetiques et son procede de preparation
WO1997028813A1 (fr) * 1996-02-06 1997-08-14 Institut Malgache De Recherches Appliquees Melanges isolables a partir de graines d'eugenia jambolana lamarck, leur preparation et l'utilisation de ces melanges et certains de leurs constituants comme medicaments
WO2000074698A1 (fr) * 1999-06-04 2000-12-14 Indian Council Of Medical Research Produit therapeutique a base de plantes
KR20020084873A (ko) * 2001-05-04 2002-11-13 알앤엘생명과학주식회사 정향피 추출물을 유효성분으로 함유하는 호르몬 대체치료용 조성물
JP2005298450A (ja) * 2004-04-15 2005-10-27 Taisho Pharmaceut Co Ltd 更年期症候群の予防又は治療剤組成物

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE CAPLUS [Online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; XP002480560 Database accession no. AN: 2007:919997 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103926108A (zh) * 2013-01-11 2014-07-16 陈军 硬组织切片取骨机

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