CHUNG YUH SHAN (CN)
LU CHAO TSEN (CN)
WANG YU TING (CN)
DCB USA LLC (US)
US20140004215A1 | 2014-01-02 |
SOKKAR, NM ET AL.: "Determination of Flavonoids in Stamen, Gynoecium, and Petals of Magnolia Grandiflora L. and their Associated Antioxidant and Hepatoprotection Activities", QUIMICA NOVA, vol. 37, no. 4, 27 March 2014 (2014-03-27), pages 667 - 671, XP055581634
CIRLINI, M ET AL.: "Phenolic and Volatile Composition of a Dry Spearmint (Mentha spicata L.) Extract", MOLECULES, vol. 21, 1007, 3 August 2016 (2016-08-03), pages 1 - 15, XP055581636
ZHU, L ET AL.: "Potent and selective inhibition of magnolol on catalytic activities of UGT1A7 and 1A9", XENOBIOTICA, vol. 42, no. 10, 16 May 2012 (2012-05-16), pages 1001 - 1008
What is claimed is: 1. A method for the preparation of a xin yi extract comprising the steps of: (a) mixing xin yi dried powder with 95% ethanol to form a suspension; (b) separating the suspension to a liquid portion and a solid portion; (c) collecting the liquid portion(s) as a crude xin yi extract; (d) subjecting the crude xin yi extract to column chromatography with macroporous adsorbent resin and eluting the column with elution solutions to obtain different eluates; (e) analyzing the eluates by High Performance Liquid Chromatography (HPLC) at the following parameters: ; and (f) collecting the eluates having four main peaks at the retention time between about 25 minutes and about 30 minutes as the xin yi extract. 2. The method of claim 1, wherein the xin yi dried powder is prepared from the flower buds of Magnolia before blooming. 3. The method of claim 2, wherein the Magnolia is Magnolia biondii, Magnolia denudate, Magnolia sprengeri, Magnolia liliflor, or combinations thereof. 4. The method of claim 1, wherein prior to step (c), the solid portion of step (b) is optionally subjected to repeat steps (a) and (b) for at least one more time. 5. The method of claim 1, wherein in step (d), the column is sequentially eluted with: (i) 3x-column volume of 60% alcohol, 4x-column volume of 80% alcohol and 4x- column volume of 95% alcohol/ethyl acetate (1 : 1); (ii) 3x-column volume of 60% ethanol, 5x-column volume of 70% alcohol and 4x- column volume of 95% alcohol/ethyl acetate (1 : 1); or (iii) 3x-coiumn volume of 60% ethanol, 4x-column volume of 95% alcohol and 4x- column volume of 95% alcohol/ethyl acetate (1 : 1). 6. A xin yi extract obtainable from the method of claim 1. 7. A xin yi extract obtainable from the method of claim 2. 8. A xin yi extract obtainable from the method of claim 3. 9. A xin yi extract obtainable from the method of claim 4. 10. A xin y i extract obtainable from the method of claim 5. 11. A pharmaceutical composition comprising a therapeutically effective amount of the xin yi extract of claim 6 and a pharmaceutically acceptable carrier. 12. A pharmaceutical composition comprising a therapeutically effective amount of the xin yi extract of claim 7 and a pharmaceutically acceptable carrier. 13. A pharmaceutical composition comprising a therapeutically effective amount of the xin yi extract of claim 8 and a pharmaceutically acceptable carrier. 14. A pharmaceutical composition comprising a therapeutically effective amount of the xin yi extract of claim 9 and a pharmaceutically acceptable carrier. 15. A pharmaceutical composition comprising a therapeutically effective amount of the xin yi extract of claim 10 and a pharmaceutically acceptable carrier. 16. A method for preventing or treating destruction and/or degeneration of alveolar bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the crude xin yi extract obtainable from the method of claim 1. 17. A method for preventing or treating destruction and/or degeneration of alveolar bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the crude xin yi extract obtainable from the method of claim 2. 18. A method for preventing or treating destruction and/or degeneration of alveolar bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the crude xin yi extract obtainable from the method of claim 3. 19. A method for preventing or treating destruction and/or degeneration of alveolar bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the crude xin yi extract obtainable from the method of claim 4. 20. A method for preventing or treating destruction and/or degeneration of alveolar bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the xin yi extract of claim 6. 21. A method for preventing or treating destruction and/or degeneration of alveolar bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the xin yi extract of claim 7. 22. A method for preventing or treating destruction and/or degeneration of alveolar bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the xin yi extract of claim 8. 23. A method for preventing or treating destruction and/or degeneration of alveolar bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the xin yi extract of claim 9. 24. A method for preventing or treating destruction and/or degeneration of alveolar bone in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the xin yi extract of claim 10. 25. A method for preventing or treating destruction and/or degeneration of alveolar bone in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 11. 26. A method for preventing or treating destruction and/or degeneration of alveolar bone in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 12. 27. A method for preventing or treating destruction and/or degeneration of alveolar bone in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 13. 28. A method for preventing or treating destruction and/or degeneration of alveolar bone in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 14. 29. A method for preventing or treating destruction and/or degeneration of alveolar bone in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 15. 30. A method for preventing or treating periodontitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the crude xin yi extract obtainable from the method of claim 1. 31. A method for preventing or treating periodontitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the crude xin yi extract obtainable from the method of claim 2. 32. A method for preventing or treating periodontitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the crude xin yi extract obtainable from the method of claim 3. 33. A method for preventing or treating periodontitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the crude xin yi extract obtainable from the method of claim 4. 34. A method for preventing or treating periodontitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the xin yi extract of claim 6. 35. A method for preventing or treating periodontitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the xin yi extract of claim 7. 36. A method for preventing or treating periodontitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the xin yi extract of claim 8. 37. A method for preventing or treating periodontitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the xin yi extract of claim 9. 38. A method for preventing or treating periodontitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the xin yi extract of claim 10. 39. A method for preventing or treating periodontitis in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 11. 40. A method for preventing or treating periodontitis in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 12. 41. A method for preventing or treating periodontitis in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 13. 42. A method for preventing or treating periodontitis in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 14. 43. A method for preventing or treating periodontitis in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 15. 44. A method for inhibiting inflammation and/or osteoclast differentiation in a subject in need thereof, comprising administering to the subj ect a therapeutically effective amount of the crude xin yi extract obtainable from the method of claim 1. 45. A method for inhibiting inflammation and/or osteoclast differentiation in a subject in need thereof, comprising administering to the subj ect a therapeutically effective amount of the crude xin yi extract obtainable from the method of claim 2. 46. A method for inhibiting inflammation and/or osteoclast differentiation in a subject in need thereof, comprising administering to the subj ect a therapeutically effective amount of the crude xin yi extract obtainable from the method of claim 3. 47. A method for inhibiting inflammation and/or osteoclast differentiation in a subject in need thereof, comprising administering to the subj ect a therapeutically effective amount of the crude xin yi extract obtainable from the method of claim 4. 48. A method for inhibiting inflammation and/or osteoclast differentiation in a subject in need thereof, comprising administering to the subj ect a therapeutically effective amount of the xin yi extract of claim 6. 49. A method for inhibiting inflammation and/or osteoclast differentiation in a subject in need thereof, comprising administering to the subj ect a therapeutically effective amount of the xin yi extract of claim 7. 50. A method for inhibiting inflammation and/or osteoclast differentiation in a subject in need thereof, comprising administering to the subj ect a therapeutically effective amount of the xin yi extract of claim 8. 51. A method for inhibiting inflammation and/or osteoclast differentiation in a subject in need thereof, comprising administering to the subj ect a therapeutically effective amount of the xin yi extract of claim 9. 52. A method for inhibiting inflammation and/or osteoclast differentiation in a subject in need thereof, comprising administering to the subj ect a therapeutically effective amount of the xin yi extract of claim 10. 53. A method for inhibiting inflammation and/or osteoclast differentiation in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 11. 54. A method for inhibiting inflammation and/or osteoclast differentiation in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 12. 55. A method for inhibiting inflammation and /or osteoclast differentiation in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 13. 56. A method for inhibiting inflammation and/or osteoclast differentiation in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 14. 57. A method for inhibiting inflammation and/or osteoclast differentiation in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 15. |
AND USE THEREOF
FIELD OF THE INVENTION
[0001] The present invention relates to the preparation of an herbal extract of Magnolia, the extract prepared therefrom, and the use of the extract in the prevention or treatment of periodontitis.
BACKGROUND OF THE INVENTION
[0002] Magnolia is a large genus of about 210 flowering plant species in the subfamily Magnolioideae of the family Magnoliaceae. In traditional Chinese herbal medicine, the bark and flower buds of Magnolia are called "hou po" and the flower buds of Magnolia are called "xin yi." Magnolia has long been used in the fields of horticulture and traditional Chinese medicine (TCM). Magnolia is reported to be effective for the treatment of rhinitis, pneumonia, bronchitis, etc. Its flowers, leaves and bark can be prepared into fragrant oils; and its branches and leaves can be distilled into essential oils. According to TCM, Magnolia can be used for the treatment of runny nose, snuffles, allergic rhinitis, eye diseases, nasal affections, body fever and chills, headache, fever in children, respiratory tract infection in children, etc. Magnolia extracts have been found to have an effect on aggregation/release of platelet and constriction of blood vessel, and thus Magnolia extracts may be a good candidate for treating cardiovascular diseases. Some references also demonstrated in animal models that Magnolia has potential in the treatment of asthma and control of the transmission of neural signals sensitive to ovalbumin.
[0003] Many publications (e.g., CA2684601C, CN102058801A, CN102240377A, CN102258573A, CN102396949A, CN102451285A, CN102698148A, CN102727856B, CN102784255A, CN102972119A, CN102972164B, CN103239700A, CN1454978A, TWI300716B, DE202010013170U1, KR717891B1, JP2008050292A, KR782599B01, CA2438055A1, AU2011241442B2, JP2005320323A, CN102188487A, CN101849991B, CN101884668B, JP7278003A, JP5212099A, JP59073584A, JP2009051740A, KR1128990B1, KR1059471B1, KR920486B1, KR883016B1, KR858438B1, KR680845B1, KR735541B1, KR2013060024A, KR2012086947A, KR2012085391A, KR2012000243 A, CN103828942A, CN103861023A, CN103598019A, CN103518544A, CN103349750A, CN102973772A, CN102835494A, CN102526441B, CN102356730A, CN102091273B, CN100998294A, CN101878900B, CN103083605A, US6280751B1, JP04488560B2, JP6128251A, JP2007319124A, JP2007314462A, JP2006225361A, JP2000178168A, KR1238825B1, KR1081059B1, KR749581B1, KR360691B, KR2009102136A, KR2009065903A, KR2008094459A, CN103898093A, CN103805442A, CN103638449A, CN103239555A, CN102078524A, CN101875802A, CN101259262B, CN100531793C, CN1840160A, CN1182863C, CN1493675A and CN201004835Y) disclose various applications oiMagnolia denudate and extracts thereof. However, none of these publications describe the efficacy of Magnolia extracts in the treatment of periodontitis.
[0004] WO 2017/061781 Al discloses a pharmaceutical composition containing ^Magnolia flower extract, a fraction thereof, or Magnolin or Fargesin as an active ingredient. WO 2017/061781 Al describes that the Magnolia flower extract and the fraction thereof can be used to promote the growth of dental roots and periodontal tissues, and thus can be used to prevent and treat periodontitis.
[0005] Most bacteria causing periodontitis are gram negative and anaerobic. For example, Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis may stick and aggregate together to form plaques. The membrane proteins of the bacteria can stimulate the cells of the host to produce pro-inflammatory factors. Therefore, such bacteria are the main cause of plaque formation leading to periodontitis. To prevent periodontitis, the primary object is to decrease the aggregation and proliferation of such pathogenic bacteria. Periodontitis is not only a main cause of tooth loss in adults older than 35, but also a factor that influences human health, such as affecting blood sugar control and increasing the incidence of heart disease, myocardial infarction, stroke, premature delivery in pregnant women, aspiration pneumonia, etc. Periodontitis is also one of the six complications of diabetes mellitus.
SUMMARY OF THE INVENTION
[0006] One aspect of the invention is to provide a method for the preparation of a xin yi active extract.
[0007] Another aspect of the invention is to provide a xin yi extract obtainable from the method of the invention.
[0008] Another aspect of the invention is to provide a pharmaceutical composition comprising the xin yi extract of the invention and a pharmaceutically acceptable carrier.
[0009] Another aspect of the invention is to provide a method for preventing or treating destruction and/or degeneration of alveolar bone of a subject in need thereof, comprising administering to the subject the extract or pharmaceutical composition of the invention.
[0010] Another aspect of the invention is to provide a method for preventing or treating periodontitis a subject in need thereof, comprising administering to the subject the extract or pharmaceutical composition of the invention. [0011] Still another aspect of the invention is to provide the use of the extract or the pharmaceutical composition of the invention in the manufacture of a medicament for preventing or treating destruction and/or degeneration of alveolar bone.
[0012] A further aspect of the invention is to provide the use of the extract or the composition of the invention in the manufacture of a medicament for preventing or treating periodontitis.
[0013] The present invention is described in detail in the following sections. Other characterizations, purposes and advantages of the present invention can be easily found in the detailed descriptions and claims of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 is an experimental design of ligature-induced periodontitis model.
[0015] Figure 2A shows the results of tomography in Control group), Ligature group and Ligature + ML-E (10 %) group in ligature-induced periodontitis model. Figure 2B shows the measured results of the distance between cemento-enamel junction CEJ and alveolar bone crest (ABC).
[0016] Figure 3A shows the CEJ and the ABC in the sliced specimens of the Control group. Figure 3B shows the the results of the sliced specimens of Control group, Ligature group and Ligature + ML-E (10 %) group in ligature-induced periodontitis model. Figure 3C shows the measured results of the distance between CEJ and ABC of the sliced specimens.
[0017] Figures 4A to 4C show the effects of the xin yi extract ML-E determined by LPS- injection animal model.
[0018] Figures 5A to 5C show the effects of the xin yi active extract mixture of ML-AF determined by LPS-injection animal model.
[0019] Figure 6 shows the HPLC fingerprint of the xin yi extract (ML-AF) obtained from the macroporous resin chromatography.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0020] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meaning commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedence over any dictionary or extrinsic definition.
[0021] Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. For example, the term "a" or "an," as used herein, is defined as one or more than one. [0022] As used herein, the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.
[0023] Often, ranges are expressed herein as from "about" one particular value and/or to "about" another particular value. When such a range is expressed, an embodiment includes the range from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the word "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to and independently of the other endpoint. As used herein the term "about" refers to ± 30%, preferably± 20%, more preferably ± 10%, and even more preferable ± 5%.
[0024] As used herein, the term "Magnolia" refers to any species of the Magnolia genus.
[0025] As used herein, the term "xin yi" refers to the flower buds ofMagnolia, and preferably, the flower buds before blooming.
[0026] As used herein, the term "extract" refers to all possible extracts that are obtained during the sample preparation process and comprise an active (lead) compound(s). The extract may be in the form of a liquid, extractum spissum, solid or powder.
[0027] As used herein, the term "active extract" refers to all possible extracts that show the desired bioactivity. Examples of such active extracts of the invention include, but are not limited to, crude extracts, column chromatographic fractions, High Performance Liquid Chromatography (HPLC)-purified fractions, etc.
[0028] As used herein, the term "organic solvent" refers to a carbon-based liquid capable of dissolving another substance.
[0029] As used herein, the term "non-polar solvent" refers to any organic solvents with a polarity index of not greater than about 2.0. Examples of such non-polar solvents include, but are not limited to, hexane, petroleum ether, carbon tetrachloride, and a mixture thereof.
[0030] As used herein, the term "polar solvent" refers to any organic solvents with a polarity index of greater than about 2.0, and generally easily miscible with water. Examples of such moderately polar solvent include, but are not limited to, methanol, ethanol, acetonitrile, and a mixture thereof.
[0031] As used herein, the term "macroporous adsorbent resin" refers to any polymer/copolymer adsorbent resin that can be an adsorbent to eliminate hydrophobic compounds, antibiotics, biomolecules, etc.
[0032] As used herein, the term "elution solution" as used herein refers to the solution that is used to elute the extract from the column chromatography, ion exchange resin, etc. [0033] As used herein, the term "preventing" or "prophylaxis" refers to delaying the onset of symptoms of a susceptible subject, reducing the occurrence of a disorder or condition, or inhibiting the occurrence of the disorder or condition, or arresting the development of the disorder or condition.
[0034] As used herein, the term "periodontitis" refers to a set of inflammatory diseases affecting the periodontium, i.e., the tissues that surround and support the teeth.
[0035] As used herein, the term "inflammatory disease" refers to lesions caused by a defensive reaction or an inflammatory reaction of a living body against the harmful influence of various circumstances (e.g. physical, chemical and biological circumstances). The classical signs of inflammatory disease are pain, heat, redness, swelling, and loss of function.
[0036] As used herein, the term "treating" or "treatment" refers to alleviating, relieving, reversing and/or improving a disorder or condition or one or more symptoms thereof, or stopping the symptoms of the disease or condition in a susceptible subject.
[0037] As used herein, the term "subject" refers to animals, especially mammals. In one preferred embodiment, the term "subject" denotes "humans."
[0038] As used herein, the term "therapeutically effective amount" refers to the amount of an active ingredient used alone or in combination with other treatments/medicaments for preventing or treating periodontitis that shows therapeutic efficacy.
[0039] As used herein, the term "pharmaceutically acceptable carrier" refers to solvents, diluents, binders, adhesives, adjuvants, excipients, acceptors, stabilizer, analogues, flavoring agents, sweetening agents, emulsifying agents or preservative agents, which are well known to persons of ordinary skill in the art, for manufacturing pharmaceutical or dietary compositions. Examples of pharmaceutically acceptable carriers include, but are not limited to, water, saline, buffers, and inert, nontoxic solids.
[0040] As used herein, the term "administering" or "administration" refers to the methods that may be used to enable delivery of the composition or medicament of the present invention to the desired site of biological action. These methods include, but are not limited to, oral, intraduodenal, nasal, parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or intradermal), topical and rectal administration.
Sources of Magnolia
[0041] Examples of Magnolia species include, but are not limited to, Magnolia biondii, Magnolia denudate, Magnolia sprengeri, Magnolia liliflor, Magnolia grandiflora, Magnolia guatemalensis, Magnolia iltisiana, Magnolia paciflca, Magnolia panamensis, Magnolia poasana, Magnolia schiedeana, Magnolia sharpii, Magnolia sororum, Magnolia tamaulipana, Magnolia virginiana, Magnolia yoroconte, Magnolia albosericea, Magnolia championii, Magnolia coco, Magnolia delavayi, Magnolia fistulosa, Magnolia henryi, Magnolia nana, Magnolia odoratissima, Magnolia pterocarpa, Magnolia gigantifolia, Magnolia hodgsonii, Magnolia lasia, Magnolia liliifera, Magnolia mariusjacobsia, Magnolia persuaveolens, Magnolia sarawakensis, Magnolia villosa, Magnolia allenii, Magnolia amazonica, Magnolia arcabucoana, Magnolia bankardiorum, Magnolia boliviano, Magnolia caricifragrans, Magnolia cespedesii, Magnolia chocoensis, Magnolia dixonii, Magnolia dodecapetala, Magnolia espinalii, Magnolia georgii, Magnolia gilbertoi, Magnolia gloriensis, Magnolia hernandezii, Magnolia irwiniana, Magnolia jardinensis, Magnolia katiorum, Magnolia lacandonica, Magnolia manguillo, Magnolia mexicana, Magnolia minor, Magnolia morii, Magnolia narinensis, Magnolia neillii, Magnolia ovata, Magnolia polyhypsophylla, Magnolia quetzel, Magnolia rimachii, Magnolia sambuensis, Magnolia santanderiana, Magnolia sellowiana, Magnolia silvioi, Magnolia venezuelensis, Magnolia virolinensis, Magnolia wolfli, Magnolia argyrothricha, Magnolia calimaensis, Magnolia calophylla, Magnolia cararensis, Magnolia chimantensis , Magnolia colombiana, Magnolia guatapensis, Magnolia jaenensis, Magnolia lenticellata, Magnolia magnifolia, Magnolia mahechae, Magnolia ptaritepuiana, Magnolia striatifolia, Magnolia urraoense, Magnolia yarumalense, Magnolia cacuminoides, Magnolia cristalensis, Magnolia cubensis, Magnolia domingensis, Magnolia ekmannii, Magnolia emarginata, Magnolia hamorii, Magnolia pallescens, Magnolia portoricensis, Magnolia splendens, Magnolia aromatica, Magnolia blaoensis, Magnolia blumei, Magnolia calophylloides, Magnolia caveana, Magnolia chevalieri, Magnolia conifera, Magnolia crassipes, Magnolia dandyi, Magnolia decidua, Magnolia dolichogyna, Magnolia duclouxii, Magnolia flglarii, Magnolia fordiana, Magnolia garrettii, Magnolia grandis, Magnolia hookeri, Magnolia insignis, Magnolia lanuginosoides, Magnolia lucida, Magnolia megaphylla, Magnolia moto, Magnolia obovalifolia, Magnolia ovoidea, Magnolia phuthoensis, Magnolia ruflbarbata, Magnolia sabahensis, Magnolia tibetica, Magnolia utilis, Magnolia ventii, Magnolia yuyuanensis, Magnolia duperreana, Magnolia kwangsiensis, Magnolia thailandica, Magnolia obovata, Magnolia officinalis, Magnolia rostrata, Magnolia tripetala, Magnolia globosa, Magnolia sieboldii, Magnolia wilsonii, Magnolia fraseri, Magnolia macrophylla, Magnolia amoena, Magnolia campbellii, Magnolia cylindrica, Magnolia dawsoniana, Magnolia denudata, Magnolia kobus, Magnolia liliiflora, Magnolia salicifolia, Magnolia sargentiana, Magnolia sprengeri, Magnolia stellata, Magnolia x soulangeana, Magnolia zenii, Magnolia loebneri, Magnolia acuminata, Magnolia x alba, Magnolia angustioblonga, Magnolia baillonii, Magnolia balansae, Magnolia banghamii, Magnolia braianensis, Magnolia cavaleriei, Magnolia champaca, Magnolia chapensis, Magnolia compressa, Magnolia coriacea, Magnolia dianica, Magnolia doltsopa, Magnolia elliptilimba, Magnolia ernestii, Magnolia flgo, Magnolia flaviflora, Magnolia floribunda, Magnolia foveolata, Magnolia fujianensis, Magnolia fulva, Magnolia guangxiensis, Magnolia hypolampra, Magnolia ingrata, Magnolia jiangxiensis, Magnolia kingii, Magnolia kisopa, Magnolia koordersiana, Magnolia lacei, Magnolia lanuginosa, Magnolia leveilleana, Magnolia macclurei, Magnolia mannii, Magnolia martinii, Magnolia masticata, Magnolia maudiae, Magnolia mediocris, Magnolia microcarpa, Magnolia microtricha, Magnolia montana, Magnolia nilagirica, Magnolia oblonga, Magnolia odora, Magnolia opipara, Magnolia philippinensis, Magnolia punduana, Magnolia rajaniana, Magnolia scortechinii, Magnolia shiluensis, Magnolia sirindhorniae, Magnolia sphaerantha, Magnolia subulifera, Magnolia sumatrae, Magnolia xanthantha, Magnolia platyphylla, Magnolia pubescens, Magnolia sulawesiana, Magnolia tsiampacca, Magnolia vrieseana, Magnolia annamensis, Magnolia carsonii, Magnolia cathcartii, Magnolia grifflthii, Magnolia gustavii, Magnolia macklottii, Magnolia pealiana, Magnolia ashtonii, Magnolia bintuluensis, Magnolia borneensis, Magnolia elegans, Magnolia pahangensis, Magnolia kachirachirai, Magnolia lotungensis, Magnolia nitida, Magnolia omeiensis, Magnolia yunnanensis , Magnolia pleiocarpa, Magnolia praecalva and Magnolia sinica.
[0042] In a preferred embodiment of the invention, the Magnolia species may include
Magnolia biondii, Magnolia denudate, Magnolia sprengeri, Magnolia liliflora and any of the combinations thereof.
The Preparation Processes and Extracts Obtainable Therefrom
[0043] The invention provides a method for extracting, isolating and purifying active extracts from the xin yi of Magnolia.
[0044] Accordingly, one aspect of this invention is to provide a method for the preparation of a xin yi extract, which comprises the steps of:
(a) mixing xin yi dried powder with 95% ethanol to form a suspension;
(b) separating the suspension to a liquid portion and a solid portion;
(c) collecting the liquid portion as a crude xin yi extract;
(d) subjecting the crude xin yi extract to column chromatography with macroporous adsorbent resin and eluting the column with elution solutions to obtain different eluates;
(e) analyzing the eluates by HPLC at the following parameters: HPLC conditions Time Solvents (%)
(min)
Injection volume: 10 · L Acetonitrile H 2 O+0.1%
Formic acid
Column: C18, 4.6 x 250 mm 0 5 95
Elution program: 0.1%FA/H 2 O ACN 10 25 75
gradient elution
20 50 50
Flow rate: 1.0 mL/min 30 55 45
Detector: UV at 254 nm 45 100 0
Column oven: 35°C 50 100 0
; and
(f) collecting the eluates having four main peaks at the retention time between about 25 minutes and about 30 minutes as the xin yi extract.
[0045] In step (b) of the present invention, any conventional method for separation can be used, such as filtration, centrifugation, or the combination thereof. For example, the suspension can be filtrated through one or more filters, and the the filter can be a Grade 1 filter paper having pore size of 11 μιτι, a Grade 2 filter paper having pore size of 8 μιτι, a Grade 3 filter paper having pore size of 6 μιτι, a Grade 4 filter paper having pore size of 20-25 μιτι, a Grade 4 filter paper having pore size of 6 μιτι, and/or a Grade 602 h filter paper having pore size of 2 μιτι.
[0046] In a preferred embodiment of the present invention, prior to step (c), the solid portion of step (b) is optionally subjected to repeat steps (a) and (b) for at least one more time.
[0047] In an embodiment of the present invention, the crude extract obtained from step (c) may be concentrated to a concentrated extract in the form of a liquid, extractum spissum, solid or powder by using any conventional concentration methods for solutions, such as vacuum concentration and rotary evaporation.
[0048] According to the present invention, the macroporous adsorbent resin used can be Diaion ® HP- 10, Diaion ® HP-20, Diaion ® HP-30, Diaion ® HP-40, Diaion ® HP-50, Amberlite ® XAD-4, Amberlite ® XAD-6, Amberlite ® XAD-7, Amberlite ® XAD-16, Amberlite ® XAD- 1180, or Amberlite ® XAD-160.
[0049] According to the invention, the elution solutions used in the column chromatography may be two or more of those known in the art such as 9: 1 to 1 : 1 of ft-hexane/ethyl acetate, 95% alcohol. [0050] According to the invention, the elution solutions used in the column chromatography may be two or more of those known in the art such as 60-95% of alcohol, 1 : 1 of alcohol/ethyl acetate.
[0051] In an embodiment of the present invention, the column is sequentially eluted with 3x- column volume of 60% ethanol, 5x-column volume of 70% alcohol and 4x-column volume of 95% alcohol /ethyl acetate (1 : 1); 3x-column volume of 60% ethanol, 4x-column volume of 80% alcohol and 4x-column volume of 95% alcohol /ethyl acetate (1 : 1); or 3x-column volume of 60% alcohol, 4x-column volume of 95% alcohol and 4x-column volume of 95% alcohol /ethyl acetate (1 : 1).
[0052] Therefore, the present invention also provides xin yi extracts obtainable from the processes described herein.
Compositions
[0053] According to one embodiment, the invention provides a composition comprising a therapeutically effective amount of a xin yi extract obtainable from the preparation method of the present invention and a pharmaceutically acceptable carrier.
[0054] In a preferred embodiment, the composition optionally comprises a conventional drug or agent useful in the prevention or treatment of periodontitis. The normal dosages of these conventional drugs or agents are well known in the art. These conventional drugs or agents include, but are not limited to, antibiotics (e.g. amoxicillin, erythromycin, cefalexin, tetracycline, and augmentin), chlorhexidine, cetylpyridinium chloride (CPC), triclosan; triclosan monophosphate, chlorhexidine, alexidine; hexetidine, sanguinarine, benzalkonium chloride, salicylanilide, domiphen bromide, tetradecylpyridinium chloride (TPC), N- tetradecyl-4-ethylpyridinium chloride (TDEPC), octenidine, delmopinol, octapinol, nisin, zinc ion agent, copper ion agent, essential oils, furanones, bacteriocins, ethyllauroyl arginate, extracts of Magnolia, a metal ion source, fluoride, stannous ions, arginine bicarbonate, honokiol, magonol, ursolic acid, ursic acid, morin, extract of sea buckthorn, a peroxide, an enzyme, a Camellia extract, a flavonoid, a flavan, halogenated diphenyl ether, creative, and propolis.
[0055] Oral compositions generally include an inert diluent or an edible carrier. Oral compositions can be liquid, or can be enclosed in gelatin capsules or compressed into tablets. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of an oral composition. Tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and/or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds typically are formulated into ointments, salves, gels, or creams as generally known in the art.
[0056] The composition can be administered to a patient orally or parenterally in the conventional forms of preparations, such as capsules, microcapsules, tablets, granules, powder, troches, pills, suppositories, injections, suspensions and syrups. Suitable formulations can be prepared by methods commonly employed using conventional, organic or inorganic carriers, such as an excipient (e.g., sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate or calcium carbonate), a binder (e.g., cellulose, methylcellulose, hydroxymethylcellulose, polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic, polyethyleneglycol, sucrose or starch), a disintegrator (e.g., starch, carboxymethylcellulose, hydroxypropylstarch, low substituted hydroxypropylcellulose, sodium bicarbonate, calcium phosphate or calcium citrate), a lubricant (e.g., magnesium stearate, light anhydrous silicic acid, talc or sodium lauryl sulfate), a flavoring agent (e.g., citric acid, menthol, glycine or orange powder), a preservative (e.g., sodium benzoate, sodium bisulfite, methylparaben or propylparaben), a stabilizer (e.g., citric acid, sodium citrate or acetic acid), a suspending agent (e.g., methylcellulose, polyvinyl pyrroliclone or aluminum stearate), a dispersing agent (e.g., hydroxypropylmethylcellulose), a diluent (e.g., water), and base wax (e.g., cocoa butter, white petrolatum or polyethylene glycol). In some embodiments, the composition of the present invention can be in the form of a semi-solid or solid such as a toothpaste, a gel dentifrice, a dental powder, a denture cleansing tablet, a chewing gum, or a solid lozenge or the like. Utility
[0057] The extract, crude extract and composition of the present invention can be used to prevent or treat destruction and/or degeneration of alveolar bone in a subject in need thereof. Therefore, the present invention provides a method for preventing or treating destruction and/or degeneration of alveolar bone in a subject in need thereof, which comprises administering to the subject the extract, crude extract and composition of the present invention.
[0058] According to the invention, the crude extract refers to the crude extract obtainable from step (d) of the method described above; and the extract refers to the xin yi extract obtainable from step (g) of the method described above. [0059] The extract, crude extract and composition of the present invention can also be used to prevent or treat periodontitis in a subject in need thereof. Therefore, the present invention further provides a method for preventing or treating periodontitis in a subject in need thereof, which comprises administering to the subject the extract, crude extract and composition of the present invention.
[0060] In another embodiment of the invention, a method is provided for inhibiting oral bacteria in a subject in need thereof, which comprises administering the extract, crude extract or composition to the oral cavity of the subject.
[0061] In a preferred embodiment, the oral bacteria include, but are not limited to,
Actinobacillus actinomycetemcomitans , Porphyromonas gingivalis, Prevotella intermedia group species, Peptostreptococcus micros, Streptococcus constellatus , Streptococcus intermedius-anginosus, Campylobacter (Wolinella) rectus, Fusobacterium nucleatum, Tannerella (Bacteroides) forsythia, Enteric gram negative rods, Enterococcus faecalis and Candida species (yeast).
[0062] In a further embodiment of the invention, a method is provided for preventing or treating oral inflammatory diseases in a subject in need thereof, which comprises administering to the oral cavity of the subject the extract, crude extract or composition to the subject.
[0063] In an alternate preferred embodiment, the extract, crude extract and composition can be optionally administered in combination with a conventional drug or agent, which is useful in the prevention or treatment of inflammatory diseases. The normal dosages of these conventional drugs or agents are well known in the art. These conventional drugs or agents include, but are not limited to, steroids or non-steroidal anti-inflammatory drug (NSAID). Examples of NSAID include, but are not limited to, salicylates (e.g. aspirin, diflunisal, salicylic acid, salsalate), propionic acid derivatives (e.g. ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen), acetic acid derivatives (e.g. indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac, nabumetone), enolic acid derivatives (e.g. piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, phenylbutazone), anthranilic acid derivatives (e.g. mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid), selective cox-2 inhibitors and sulfonanilides (e.g. nimesulide).
[0064] The invention further provides a method for inhibiting osteoclast differentiation in a subject in need thereof, which comprises administering the extract, crude extract or composition to a subject. [0065] In an alternate preferred embodiment, a conventional drug or agent used in the inhibition of osteoclast differentiation is co-administered. The normal dosages of these conventional drugs or agents are well known in the art. These conventional drugs or agents include but are not limited to antiresoptive agent (e.g. bisphosphonate, receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitor, selective estrogen receptor modulator (SERMs) such as clomifene, ormeloxifene, raloxifene, tamoxifen, toremifene, lasofoxifene and ospemifene), anabolic agent (e.g. teriparatide) and strontium ranelate.
[0066] The invention further provides a method for inhibiting inflammation in a subj ect in need thereof, which comprises administering the extract, crude extract or composition to a subject.
[0067] The following examples are provided to aid those skilled in the art in practicing the present invention. Even so, the examples should not be construed to unduly limit the present invention as modifications to and variations on the embodiments discussed herein may be made by those having ordinary skill in the art without departing from the spirit or scope of the present inventive discovery.
Example 1: Preparation of Xin Yi Extract (ML-E)
[0068] 222.0 g of dry xin yi material was ground to obtain 212.0 g xin yi powder. The xin yi powder was soaked and shaken in lOx volume of 95% ethanol (w:v = 1 : 10) for 30 minutes and then kept soaking for one day. The mixture was filtrated through a No.1 filter to obtain a first filtrate. The solid residue was again soaked in a 1 Ox-volume 95% ethanol (w:v = 1 : 10) overnight. The mixture was filtrated through a No.1 filter to obtain a second filtrate. The two filtrates were combined and concentrated under reduced pressure to obtain an extractum spissum (ML-E) with a yield of 10-14%.
Example 2: Treatment Effect of ML-E in Periodontitis Model Induced by Ligature
Step 1 : Establishment of Ligature-Induced Periodontitis Model
[0069] Periodontitis is a prevalent oral inflammatory disease that leads to alveolar bone loss and may exert an adverse impact on systemic health. Ligature-induced periodontitis model has been used frequently in relatively large animals, including non-human primates, to assess the host response and its effects on the tooth-supporting tissues (gingiva and bone) under well- controlled conditions (see Toshiharu Abe and Georage Hajishengallis; J Immunol Methods. 2013 August 30; 394(0): 49-54).
[0070] In this example, adult male Sprague Dawley (SD) rats having a weight ranging from about 280 to 310 grams were used. The method for the generation of ligature-induced periodontitis was previously reported in Cai et al. (J Periodontal Res. 2008 Feb; 43(1): 14-21). Briefly, the rats were anesthetized by intraperitoneal injection and a nylon thread was subjected around the necks of both first molars of mandible and the necks of both second molars of the palate. The ligature was also placed on the mesial surface of buccal side. The experimental design is shown in Fig. 1. The rats were sacrificed on Day 11 and the variation of alveolar bone height was observed by tomography (Cheng et al, J Periodont Res, 2010; 45: 788-795). Step 2: Measurement of the Height of Alveolar Bone
[0071] The soft tissues of the mandible of the rats were removed and the position of cemento- enamelj unction (CEJ) was identifiedcd. The distance between the CEJ and the CEJ-alveolar bone crest (ABC) was measured by tomography so as to observe the variation of the height of the alveolar bone.
Example 3: Effect of Xin Yi Extract (ML-E) Determined by Ligature-Induced Periodontitis Animal Model
[0072] This example demonstrates that xin yi extract (ML-E) can decrease the destruction/degeneration of alveolar bone in ligature-induced periodontitis animal model. The animals were randomized into 3 groups, i.e., Control group; Ligature group; and Ligature + ML-E (10 %) group
[0073] The tomography results (see Figs. 2A and 2B) reveal that the animals of ligature group showed severe bone matrix absorption, but the treatment of ML-E could significantly prevent destruction/degeneration of alveolar bone at the interdental site in the animals of group by the treatment with ML-E (10%).
[0074] The results of the sliced specimens from the animals are shown in Figs. 3Ato 3C. Fig. 3A shows the normal distance between CEJ and ABC. According to Figs. 3B and 3C, the animals of liateure group experienced severe bone matrix absorption but the destruction/degeneration of alveolar bone at the interdental site of the animals was significantly inhibitted by the treatment of ML-E.
Example 4: Xin Yi Active Extract (ML-AF) Prepared by Using Macroporous Resin
[0075] Step 1 : About 15 L of macroporous resin DIAION ® HP20 was soaked in 95% ethanol ("soaked solution") overnight. The resin was washed with ddFhO twice to remove ethanol and then soaked in 60% ethanol ("alcohol solution"). The resin was filled into a reverse phase chromatography column and washed by 45 L of 60% ethanol. The resin was packed tightly to ensure that there were no bubble in the column. [0076] Step 2: 504 g of ML-E were dissolved in 750 mL of ethanol and then added to DIAION® HP20 resin. The contents (including the extract and the resin) were drained by a concentrator to obtain a dry powder.
[0077] Step 3 : The sample was poured into a column and eluted with 3x-column volume of 60% ethanol, 4x-column volume of 80% ethanol and 4x-column volume of 95% ethanol/ethyl acetate (1 : 1), until the color of the eluates remained unchanged.
[0078] Step 4: All the eluted fractions were examined by HPLC and the active fractions were collected in an 80% alcohol solution.
[0079] Step 5 : The obtained alcohol solution was concentrated to obtain a xin yi active extract (ML-AF) with a yield of 4.07%.
Example 5 : Anti-Inflammatory Activity and Osteocleastgenesis inhibition of ML-E and a Xin Yi Active Extract (ML-AF)
[0080] In this experiment, NO production inhibition assay and RANKL-induced osteoclastogenesis were used to test the xin yi crude extract of (ML-E) and a xin yi active extract mixture of ML-AF.
Assay of nitrite production
[0081] RAW 264.7 cells were seeded in a 96-well plate (3 χ 10 4 cells/ well) in a basal medium. After incubating for 24 h, cells were treated with a lipopolysaccaride (LPS) (100 ng/mL) and an indicated concentration of xin yi extract according to a modification of the method disclosed in Molecules., 2010, 15, 7815-7824. The nitrite concentrations of the supernatants were determined by using a Griess reagent kit after 24 h. Cell proliferation was evaluated by a cell growth inhibitory assay using MTS reagent.
Determination of osteoclast differentiation
[0082] RAW 264.7 cell were seeded in a 96-well plate (10 3 cells/ well) in a basal medium. After incubating for 24 h, the medium was replaced with a differentiation medium containing 50 ng/mL RANKL and an indicated concentration of xin yi extract. After 4 days, the cellular enzymatic activity of tartrate-resistant acid phosphatase (TRAP), a marker enzyme of osteoclasts, was measured according to a modification of the method disclosed in Joumal of Biological Chemistry, 2004, 279, 13984-13992. Briefly, the medium was removed and the cells was gently washed twice with PBS. The cells were fixed in 10% formalin for 10 min and washed in 95% ethanol, and then incubated in 0.1 mL of phosphatase substrate (3.7 mM p- nitrophenyl phosphate in 50 mM citrate buffer pH4.6) in the presence of 10 mM sodium tartrate at 25°C for 30 min. After incubation, the reaction mixtures were transferred to a new well plate containing an equal volume of 0.1 N NaOH and the absorbance of each well was measured at 405 nm using Bio-Rad Model 680 microplate reader.
[0083] The data are shown in Table 1 and Table 2 below.
Table 1 : Assay of Nitrite Production
Table 2: Determination of Osteoclast Differentiation
: Inhibition rate were compared to RANKL-alone treatment
Example 6: Effects of Xin Yi Extract (ML-E) and Xin Yi Active Extract Mixture of ML- AF Determined by LPS-injection Animal Model
[0084] In this example, adult male Sprague Dawley (SD) rats having a weight ranging from about 280 to 310 grams were used. The animals were randomized into 3 groups, i.e., Group 1 (PBS, Control group); Group 2 (LPS); Group 3 (LPS +test sample) (test sample : 10 % ML-E and 10% ML-AF. The method for the generation of an LPS-induced osteoclast formation was previously reported in Cheng et al. (J Periodont Res, 2010; 45: 788-795). ML-E and xin yi active extract mixture (ML-AF) were respectively applied to the affected area of each rat on Day 1 to Day 8, and 5 mg/mL LPS were injected into the right jaw site (10 μί/βίίβ) of the rats on Day 2 to Day 4. The rats were sacrificed on Day 8 and the numbers of osteoclasts were counted by histochemical staining. [0085] The results are shown in Figures 4A to 4C and 5A to 5C. Data are mean ± SD of 4-5 rats per group and statistically analyzed by Student's t-test. In summary, LPS significantly induced osteoclast differentiation and eroded alveolar bone surface in Group 2. However, administration of ML-E (10 %) as well as ML-AF (10 %) effectively reduced osteoclast differentiation and reduced the erosion of alveolar bone surface.
Example 7: High Performance Liquid Chromatography (HPLC) Fingerprint of Xin Yi Active Extracts (ML-AF)
[0086] In this experiment, HPLC was used to analyze the components in the xin yi active extract mixture (ML-AF). The parameters used for HPLC are listed in Table 3 below.
Table 3
[0087] The HPLC fingerprint for ML-AF obtained from the macroporous resin chromatography is shown in Fig 6. Four main peaks were found at the retention times of 26.3, 26.9, 27.6, and 28.7 minutes in the xin yi active extracts mixture (ML-AF).
[0088] Numerous modifications and variations of the invention as set forth in the above illustrative examples are expected to occur to those skilled in the art. Consequently, only such limitations as appear in the appended claims should be placed on the invention.