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WO2008004118A2 - Extraits de carthamus tinctoris pour le traitement de l'ostéoporose, et leur procédé d'extraction - Google Patents

Extraits de carthamus tinctoris pour le traitement de l'ostéoporose, et leur procédé d'extraction Download PDF

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Publication number
WO2008004118A2
WO2008004118A2 PCT/IB2007/002062 IB2007002062W WO2008004118A2 WO 2008004118 A2 WO2008004118 A2 WO 2008004118A2 IB 2007002062 W IB2007002062 W IB 2007002062W WO 2008004118 A2 WO2008004118 A2 WO 2008004118A2
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Prior art keywords
extract
extracts
bone
osteoporosis
carthamus
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PCT/IB2007/002062
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WO2008004118A3 (fr
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Villoo Morawala Patell
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Avestha Gengraine Technologies Pvt. Ltd.
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Publication of WO2008004118A2 publication Critical patent/WO2008004118A2/fr
Publication of WO2008004118A3 publication Critical patent/WO2008004118A3/fr

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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)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/286—Carthamus (distaff thistle)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis

Definitions

  • the invention relates to compositions and methods for preventing, treating, or managing osteoporosis o ⁇ 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 Carthamus tinctoris 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 Carthamus tinctoris 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 stem of Carthamus tinctoris 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
  • osteoporosis is categorized as a primary or secondary disease.
  • the latter 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.
  • BMD bone mineral density
  • DEXA dual energy x-ray absorptiometry
  • 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).
  • 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 systemic 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 ⁇ n addition to drug therapy
  • 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 characterised by high bone resorption. Selective estrogen receptor modulators (SERMS) mimic estrogens in some tissues and anti-estrogens in. others, and ideally provide the bone-retaining effects of estrogen without its unwanted side effects.
  • SERM selective estrogen receptor modulators
  • raloxifene the only marketed SERM is raloxifene. Raloxifene prevents bone loss and is indicated for the prevention and treatment of vertebral fractures in postmenopausal women.
  • 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.
  • safflower seeds have beneficial effects on various bone diseases such as bone fracture, osteoporosis, and osteodysplasia. In addition, they are known to have anti-inflammatory effects.
  • Safflower (Carthamus tinctorius L.) seeds have long been clinically used in Korea to promote bone formation and prevent osteoporosis,
  • the beneficial effect of safflower seeds may be mediated, at least in part, by the stimulating effect of polyphenols compounds on proliferation of osteoblasts.
  • Kim et al evaluated the effect of a safflower seed extract on the regeneration of periodontal tissue in a preclinical 1-wall model in dogs. They reported that root resorption was often observed, but no ankylosis was present. Wound conditioning with safflower seed extracts may contribute to bone formation but appears to have unpredictable potential for stimulating periodontal regeneration including new cementum, Hong et al investigated that water extracts of Carthamus inhibited IL-I beta- induced bone resorption.
  • 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 Carthamus tinctoris 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 stems of Carthamus tinctoris 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 Carthamus tinct ⁇ ris plant stems. The process comprises of:
  • 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 Carthamus tinctoris as described above, by studying their performance in cell based assay models.
  • Fig 1 The cell viability for extract AN05 : AN06 and ANl 1
  • FIG 2 The CTX release for plant extract AN05, AN06 and ANl 1 for the resorption experiment
  • Fig 3 The cell viability for plant extracts AN05, AN06 and ANI l for the resorption experiment
  • Fig 4 The CTX release for plant extract AN05 lead characterization
  • Fig 5 The CTX release for plant extract ANQ6 lead characterization
  • Fig 6 The ceil viability for plant extract AN05 lead characterization
  • Fig 7 The cell viability for plant extract ANQ6 lead characterization
  • Fig 9 The TRAP activity for plant extract AN06 lead characterization
  • Fig 10 The ALP activity for plant extract AN05 and AN06 for the bone formation experiment
  • Fig 11 The ALP activity for plant extract ANl 1 for the bone formation experiment
  • Fig 12 The cell viability for plant extracts AN05 and AN06 for the bone formation experiment
  • Fig 14 The cell viability for plant extracts ANl 1 for the bone formation experiment
  • 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 Carthamus tinctoris as defined herein.
  • the mammal is a human being.
  • "treating a disease" in a mammal 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 Qt becoming more invasive, or slowing down the rate of advance of a disease.
  • a prophylactic method for preventing the occurrence of a disease state in a mammal which comprises administering to the said mammal an effective non-toxic amount of an extract from Carthamus tinctoris as defined herein in the preparation of a comestible ( ⁇ foodstuff) for prophylaxis against the occurrence of Osteoporosis.
  • the mammal is human and the said extract comprises a single extract from a plant part of Carthamus tinctoris or a combination of extracts therefrom as detailed herein.
  • the present invention further relates to extracts, which may be isolated from stems of the Carthamus tinctoris 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 stems of Carthamus tinctoris, 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 Carthamus tinctoris plant, depending on the end purpose that is required of the extract.
  • the choice of selected plant material may be of any type but is preferably the stems of the Carthamus tinctoris 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 colorimetric 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- dxug 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(1): 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 etal; 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 CDH positive monocytes have been selected for osteoclastogenesis. (Clin.Sc. (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 boae 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.
  • Organ cultures rely upon the release in vitro of Ca from long bones or calvaria from newborn mice or rats (Raisz, 1963; van der Pluijm et al. 1994; Most et al. 1995).
  • the bones are pre-labeled by incorporation into the pups of 45Ca or [3H] proline injected into the pregnant mother.
  • the release of the isotopes from the cultured bones is measured at the end of the experiment, which can be continued for at least 5 days. This assay has been used frequently, but the cell composition of explants is extremely heterogeneous.
  • Bone particle-based assays Bone particles, obtained by grinding of 45Ca- or [3H] pro ⁇ ine-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 (B ⁇ yde et all 984) 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, Parf.tU995; 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-gylceroJ 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)..
  • One of the most frequently assayed biochemical markers is alkaline phosphatase, which is simple to measure biochemically.
  • 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 Carthamus tinctoris, in admixture with a pharmaceutically acceptable carrier, Preferably, 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 o ⁇ Carthamus substantially devoid of undesirable contaminating compounds, The plant extracts may have, for procedure, undergone a number of solvent extraction steps substantially to separate out undesirable components from desirable components such as those alluded to in the procedures 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 booe 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.
  • 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 carriers 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 I 3 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 Carthamus species.
  • a pharmaceutical formulation may contain more than one active extract derived from two or more Carthamus 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.
  • Bmulgents 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, hi 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 acaoia 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, bindeis, surface active agents, thickeners, lubricants, preservatives (including antioxidants) and the like,
  • ⁇ he 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. It is thought that a similar multi-functional role could also 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 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 fruit bars, biscuits, dairy products such as yoghurts, milk and milk-based products such as custards, cream, cheese, butter and creme fraiehe.
  • simulated dairy food products such as margarine, olive oil-based spreads, and low fat cream substitutes such as Elmlea products, fruit 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, fruited 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, iced yoghurts and the like.
  • 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 fruited oils
  • fruited oils such as sunflower oil, rapeseed oil, olive oil,, walnut, hazelnut, and sesame seed oil and the like
  • 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 r ⁇ g twice daily.
  • Extraction of Carthamus tinctoris stem was carried out by direct extraction method, at 20 - 85 C in reflux extractor apparatus 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).
  • Various solvents used for extraction are Hexane, 80% Ethanol and Acetone.
  • 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.
  • 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.
  • 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.
  • AN05 was toxic at the dilution 1 :100, but it was not toxic at dilution 1:1000 and 1:10,000.
  • AN06 and ANI l were not toxic at any of the dilutions. Dilution 1:1000 and 1:10,000 will be used for the hit screening in the bone resorption assay and in the bone formation assay, because these dilutions are not toxic to any of the extracts.
  • 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 AN05 which is Carthamus tinctoris extract made with 80% ethanol, is not toxic at either dilution. However, the extract has an inhibitory effect on the bone resorption both at dilution 1 :1000 and also at dilution 1 :10,000. AN05 inhibit bone resorption more than the positive control, Bafilomycin. The extract inhibits bone resorption ⁇ 95% compared to the control at 1:1000 and ⁇ 60% compared to the control at 1:10,000.
  • Plant extract AN06 which is Carthamus tinctoris extract made with acetone, is not toxic at either dilution. However, the extract has an inhibitory effect on the bone resorption at dilution 1 : 1000. AN06 inhibit the bone resorption -75% compared to the control. Plant extract ANl 1, which is Carthamus tinctoris extract made with hexane, is not toxic at either concentration. The extract inhibits bone resorption at dilution 1 :1000 ⁇ 60% compared to the control.
  • Extract AN05 and AN06 were selected for further test in bone resorption lead characterisation. Extract ANl 1 was not selected even though it inhibit resorption ⁇ 60% compared to the control, because AN05 and AN06 inhibit bone resorption to a much lower level.
  • AN06 inhibited resorption ⁇ 50 % compared to the control at dilution 1:1000 in the lead characterisation, while it inhibited resorption ⁇ 75 % compared to the control at dilution 1:1000 in the hit screening. The difference is due to variations in the bone resorption experiment.
  • 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.
  • BMP-2 used as a positive control, while treatment without ascorbic acid and ⁇ -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.

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  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Rheumatology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Medical Informatics (AREA)
  • Botany (AREA)
  • Biotechnology (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract

L'invention porte sur des compositions et méthodes de prévention, traitement, et gestion de l'ostéoporose ou d'états caractérisés par une résorption osseuse accrue, consistant à administrer une dose à effet prophylactique et thérapeutique de Carthamus tinctoris ou de ses extraits, à un mammifère de préférence humain, le nécessitant. Les compositions peuvent comprendre un seul ou plusieurs extraits. L'invention porte également sur des extraits isolés de différentes parties de la Carthamus tinctoris, sur leur préparation, sur des médicaments les contenant, sur l'utilisation de ces extraits et des constituants nécessaires à leur préparation, ainsi que leur procédé de préparation à partir de différentes parties de la Carthamus tinctoris.
PCT/IB2007/002062 2006-07-06 2007-07-09 Extraits de carthamus tinctoris pour le traitement de l'ostéoporose, et leur procédé d'extraction WO2008004118A2 (fr)

Applications Claiming Priority (2)

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IN1176CH2006 2006-07-06
IN1176/CHE/2006 2006-07-06

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WO2008004118A2 true WO2008004118A2 (fr) 2008-01-10
WO2008004118A3 WO2008004118A3 (fr) 2008-06-05

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102552460A (zh) * 2012-02-10 2012-07-11 河南省正骨研究院 一种益气生骨中药制剂及其制备方法
CN103816197A (zh) * 2012-11-16 2014-05-28 邢秋苓 一种抑制破骨细胞形成的红花提取物
CN103816198A (zh) * 2012-11-16 2014-05-28 邢秋苓 一种红花提取物及其应用
CN105079725A (zh) * 2015-10-08 2015-11-25 王磊 一种治疗骨折中药组合物
CN105106363A (zh) * 2015-09-29 2015-12-02 王磊 一种促进骨伤骨折愈合的中药

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100354791B1 (ko) * 2000-04-04 2002-10-05 최상원 홍화씨에서 추출한 폴리페놀화합물의 신규한 용도
KR100391801B1 (ko) * 2000-10-12 2003-07-16 김도완 홍화씨 분말을 함유하는 건강기능성 스낵 및 그 제조방법
KR20030038870A (ko) * 2001-11-06 2003-05-17 경상북도(승계청:경상북도농업기술원,관리청:경상북도 도지사) 볶은 홍화씨 미세분말을 첨가한 양갱의 제조방법
EP1325681A1 (fr) * 2001-12-11 2003-07-09 Société des Produits Nestlé S.A. Composition pour la promotion de la croissance osseuse et la conservation de la santé osseuse
US7195790B2 (en) * 2002-12-09 2007-03-27 Shaklee Corporation Modification of cyclooxygenase and lipoxygenase activity with asteridae extracts and optionally boswellic acid
KR20030044973A (ko) * 2003-05-16 2003-06-09 황규현 신경통, 관절염, 골다공증 예방식품 제조방법
KR20050050291A (ko) * 2003-11-25 2005-05-31 이광현 한약 치료제

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102552460A (zh) * 2012-02-10 2012-07-11 河南省正骨研究院 一种益气生骨中药制剂及其制备方法
CN103816197A (zh) * 2012-11-16 2014-05-28 邢秋苓 一种抑制破骨细胞形成的红花提取物
CN103816198A (zh) * 2012-11-16 2014-05-28 邢秋苓 一种红花提取物及其应用
CN105106363A (zh) * 2015-09-29 2015-12-02 王磊 一种促进骨伤骨折愈合的中药
CN105079725A (zh) * 2015-10-08 2015-11-25 王磊 一种治疗骨折中药组合物

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