WO2011034447A1 - Composition active obtenue à partir de bois de cervidé et ses procédés d'utilisation - Google Patents

Composition active obtenue à partir de bois de cervidé et ses procédés d'utilisation Download PDF

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Publication number
WO2011034447A1
WO2011034447A1 PCT/NZ2010/000185 NZ2010000185W WO2011034447A1 WO 2011034447 A1 WO2011034447 A1 WO 2011034447A1 NZ 2010000185 W NZ2010000185 W NZ 2010000185W WO 2011034447 A1 WO2011034447 A1 WO 2011034447A1
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WO
WIPO (PCT)
Prior art keywords
hard
antler
cell function
harvested
deer antler
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PCT/NZ2010/000185
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English (en)
Inventor
David Collie
James Christopher Anderson
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Andeer (Nz) Limited
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Publication of WO2011034447A1 publication Critical patent/WO2011034447A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane

Definitions

  • the invention relates to an active composition and methods of use thereof. More specifically, the invention relates to a harvested hard deer antler which increases osteoblast cell activity and also decreases / inhibits osteoclast cell activity.
  • the composition may be useful in treatment of bone diseases such as osteoporosis and in treament of bone fractures.
  • Osteoporosis is defined as a depletion of bone mass compounded with micro-architectural deterioration of bone tissue leading to increased fragility and therefore higher susceptibility to fracture. Increased bone resorption coupled with a reduced rate of new bone formation to replace the lost bone leads to decreased bone mineral density and mass and is characteristic of secondary osteoporosis. Osteoporosis affects a significant number of people worldwide with one in three women and one in five men being at risk of osteoporosis.
  • osteoporosis the cell responsible for bone resorption
  • BMD bone mineral density
  • Long-term suppression of bone resorption has been ineffective in bone remodelling.
  • the use of growth factors, hormone therapy and fluoride compounds, to stimulate bone formation are limited in their application. Safe and effective treatment for osteoporosis should aim at decreasing osteoclast activity and hence bone resorption and facilitate bone repair.
  • bone diseases are also associated with changes in normal osteogenic activity.
  • bone healing post an injury also relies (at least in part) on osteogenic effects such as osteoblast cell function hence inhibiting osteoclast cell development and/or stimulating osteoblast cell proliferation may have many applications around bone health.
  • the deer antler is highly vascular tissue and part of this means that antler grows rapidly and tissue damage regenerates rapidly.
  • the rapid growth of this bony structure is due to the action of large numbers of osteoblasts as well as the hematopoetically derived osteoclasts progenitors circulating in the complex blood supply of the antlers.
  • the antler tips grow at a rapid rate, associated with extensive osteoclastic resorption of mineralised cartilage from matrix.
  • IGF-1 Insulin like growth factor
  • the role of parathyroid hormone in bone formation is evident by increased bone sensitivity to PTH in ovariectomized rats. Deer antlers undergo regeneration annually. This phenomenon has been attributed to the presence of nucleus derived, parathyroid hormone - related protein receptor (PTHrP) that influences osteoclasts differentiation and the regulation of skeletal tissue regeneration.
  • Deer antler has been a traditional oriental medicine. In the past, the emphasis of almost all research has been on use of soft antler or velvet antler and hence the cost of velvet is significantly higher than that of hard antler. There is extensive art about various uses of velvet including for treatment of bone disease in humans. Less well studied is the use of hard antler for treating bone ailments. The studies that have been completed are of cast antler. Harvesting hard antler from the deer before casting but after stripping and transformation to a mineralised form is relatively unusual as most deer are not farmed and instead are wild. The New Zealand deer industry is largely made up of farmed deer where harvesting of antler at different times in the antler lifecycle is both practical and economically viable.
  • the invention broadly relates to harvested hard deer antler powders and extracts and various methods and uses of the harvested hard deer antler around inhibiting osteoclast cell development and also increasing osteoblast cell function.
  • the harvested hard deer antler has an unexpectedly useful osteogenic activity compared to cast hard antler and traditional remedies using velvet.
  • a formulation including hard deer antler that, on administration to an animal, (a) increases osteoblast cell function; or
  • hard deer antler is harvested from deer in the time period between stripping and one month before casting.
  • Figure 1 illustrates the peak activity for velvet, harvested hard antler and cast antler.
  • Figure 2 illustrates the effect of the cast antler extract on RAW 264.7 osteoclastogenesis.
  • FIG. 3 illustrates the effect of the harvested hard antler extract on RAW 264.7
  • Figure 4 illustrates the effect of the harvested hard deer antler pre-treated with an ethanol wash on RAW 264.7 osteoclastogenesis. Significance (p ⁇ 0.05) from the 0 control is indicated by an asterisk. Means ⁇ 95% CI; Figure 5 illustrates the effect of the velvet extract on RAW 264.7 osteoclastogenesis.
  • Figure 6 illustrates the composite graphs showing the effect of harvested hard antler and velvet powdered fractions on RAW 264.7 osteoclastogenesis. Significance (p ⁇ 0.05) from the 0 control is indicated by an asterisk. Means ⁇ 95% CI; and
  • Figure 7 illustrates the effect of harvested hard antler and velvet powdered fractions on osteoblast proliferation. Significance (p ⁇ 0.05) from the 0 control is indicated by an asterisk. Means ⁇ 95% CI.
  • the invention broadly relates to harvested hard deer antler powders and extracts and various methods and uses of the harvested hard deer antler around inhibiting osteoclast cell development and also increasing osteoblast cell function.
  • the harvested hard deer antler has an unexpectedly useful osteogenic activity compared to cast hard antler and traditional remedies using velvet.
  • 'hard deer antler' refers to calcified deer antler post stripping.
  • 'harvested hard deer antler 1 - refers to deer antler being removed e.g. by cutting, from the deer in the time period between stripping and approximately one month prior to natural casting.
  • 'stripping' refers to the time period characterised by a rapid increase in antler mineral content and stripping of the soft outer covering of the antler.
  • this stripping stage typically in the autumn season
  • the stag rubs the antler in shrubs and trees and as a result, the antler(s) become hard and polished.
  • 'casting' refers to the natural process where the hard calcified antler naturally drops from the deer, typically in the spring season.
  • a formulation including hard deer antler that, on administration to an animal,
  • the hard deer antler is harvested from deer in the time period between stripping and one month before casting.
  • the harvested hard deer antler formulation of the embodiment above may be characterised by a double action on administration of increasing osteoblast cell function and decreasing osteoclast cell function.
  • the hard deer antler may have an ash content of greater than or equal to 26% by weight. More preferably, the ash content may be greater than or equal to 35% by weight. Still more preferably, the ash content may be greater than 50% by weight.
  • the hard deer antler may have a protein content of greater than or equal to 30-50% by weight. More preferably, the protein content is greater than 50% by weight.
  • the hard deer antler is in the form of a powder. This should not be seen as limiting as other forms may be used for example a tonic or liquid elixir.
  • the powder hard antler may be micronised.
  • the powder size may be less than approximately 150 micron. More preferably, the powder may be less than 75 micron.
  • the powder may be administered to a subject by known methods such as oral or parenteral methods.
  • a preferred method is likely to be via oral administration due to ease of administration although this should not be seen as limiting.
  • the amount administered may vary from 5 to 500 mg hard deer antler per day per kg bodyweight.
  • the amount administered varies from 20 to 100 mg hard deer antler per day per kg bodyweight.
  • the dosage may be approximately 50 mg hard deer antler per day per kg bodyweight.
  • the powder may also increase the mineral content and strengt of bones.
  • the inventors understand that this effect may be a function at least in part of the increased osteoblast cell activity and decreased osteoclast cell activity.
  • the effect may also in part be due to the fact that the hard antler powder also includes minerals present in the antler such as calcium.
  • the minerals, and calcium are provided in the form of microcrystalline hydroyapatite - a form known in bone supplements to be ideal for absorption as the calcium and other nutrients are easily accessed and absorbed by the body.
  • alternative calcium sources such as calcium carbonate are poorly absorbed by the body as the calcium needs to be. processed by the body in order to be absorbed. Side effects associated with alternative calcium sources are also avoided. For example, some users of calcium carbonate supplements report bloating which may be a consequence of the carbonate forming carbon dioxide gas in the body.
  • the formulation may be produced by drying the harvested hard antler and then extracting / concentrating the dried protein fraction of the antler into a powder.
  • the extraction may reduce the mineral and liquid fractions to less than 10% weight of the formulations.
  • the moisture content of the hard antler before drying is typically 10 to 50% by weight. After drying, the residual water content may less than 10% by weight.
  • the protein . fraction may be extracted via an aqueous extraction method.
  • the resulting extract may then be dried and optionally ground into a powder. This extract may have a greater activity due to the bioactive(s) responsible for the osteogenic activity being in greater concentration or in a profile , that provides a synergy.
  • the formulation may be produced by drying the harvested hard antler and grinding the dried antler into a powder.
  • this formulation includes the mineral and lipid fractions as well as protein fraction.
  • the harvested hard deer antler may be produced by manufacturing a powder from the raw harvested hard deer antler which may then be washed using alcohol and the resulting washed product passed through a separation process to remove the alcohol.
  • the resulting alcohol washed product may then either be used as is or further extracted, for example, via an aqueous extraction process.
  • an alcohol wash may not be desirable as this unexpectedly removes osteoblast proliferation observed for harvested hard deer antler that is not washed with alcohol. Nevertheless, osteoclast inhibition remains irrespective of an alcohol wash and therefore, for certain applications, an alcohol wash may have a purpose.
  • an alcohol wash such as with ethanol, is common in the art in order to reduce microbial loading.
  • the harvested hard deer antler formulation of the embodiment above may be characterised by a double action on administration of increasing osteoblast cell function and decreasing osteoclast cell function.
  • the harvested hard deer antler may undergo an extraction process to reduce the mineral and lipid fractions to less than 10% weight of the formulation.
  • the extraction process may be an aqueous based extraction process.
  • the harvested hard deer antler used in the second embodiment is dried and ground to a powder.
  • this method retains the mineral and lipid fractions as well as the protein fraction of the harvested hard deer antler.
  • the inventors envisage that the powder produced by the method of the second embodiment may be administered by known methods such as oral or parenteral methods. A preferred method is likely to be via oral administration due to ease of administration although this should not be seen as limiting.
  • the amount administered according to the method of the second embodiment may vary from 5 to 500 mg powdered hard deer antler per day per kg bodyweight.
  • the amount administered varies from 20 to 100 mg powdered hard deer antler per day per kg bodyweight.
  • the dosage may be approximately 50 mg powdered hard deer antler per day per kg bodyweight.
  • the animal may be a human although non-humans such as horses, dogs, cats, livestock and so on may also be treated.
  • the disease or condition maybe osteoporosis.
  • the condition may be one or more bone fractures.
  • the hard deer antler formulation of the embodiment above may be characterised by a double action on administration of increasing osteoblast cell function and decreasing osteoclast cell function.
  • the powered hard deer antler may undergo an extraction process to reduce the mineral and lipid fractions to less than 10% weight of the formulation.
  • the extraction process may be an aqueous based extraction process.
  • the harvested hard deer antler used in the second embodiment is dried and ground to a powder.
  • this method retains the mineral and lipid fractions as well as the protein fraction of the harvested hard deer antler.
  • the hard deer antler of the third embodiment may be administered by known methods such as oral or parenteral methods.
  • a preferred method is likely to be via oral administration due to ease of administration although this should not be seen as limiting.
  • the amount administered according to the third embodiment may vary from 5 to 500 mg powdered hard deer antler per day per kg bodyweight.
  • the amount administered varies from 20 to 100 mg powdered hard deer antler per day per kg bodyweight. Note this may vary as discussed above.
  • the animal may be a human although non-humans such as horses, dogs, cats, livestock and so on may also be treated.
  • the disease or condition maybe osteoporosis.
  • the condition may be one or more bone fractures.
  • the above formulations and associated methods and uses may have a variety of potential applications, particularly in treatment of osteoporosis and other bone diseases, as well as in recovery from bone fractures. It is acknowledged that, although deer velvet is known to assist in treatment of bone disease, hard deer antler is less well studied and certainly not hard antler that has been harvested from the deer prior to casting. The natural lifecycle of the antler means that the exact composition of the antler changes significantly during the various parts of the cycle and hence the bioactives and their concentrations vary considerably as well.
  • a main advantage for the powder may be a potential double effect by encouraging calcium deposition in the bone as well as inhibiting calcium removal.
  • a further advantage of the invention formulation over the art is that, as the powder/extract is manufactured from hard antler, the cost of sourcing the raw material is significantly lower than using velvet.
  • unwanted chemical treatments may be avoided such as use of alcohols in extraction and/or pre-treatment washing and the steps required to remove such alcohols from the extracts may also be avoided. This change also reduces processing costs and potentially other aspects of the extract such as avoiding the bitter taste can be present in alcohol extracted materials.
  • alcohol washing techniques may be used in selected circumstances and hence are not excluded from the invention.
  • the powder potentially provide osteoblast and osteoclast effects, but the powder also provides important minerals to health and in particular calcium.
  • the calcium is provided as microcrystalline hydroxyapatite, the bioavailability of the calcium from the powder is very high hence the powder provides both cell effects but a potent calcium source as well.
  • each group was fed a low calcium diet (0.1 % calcium) for 4 weeks (28 days) to exacerbate ovariectomy induced bone-loss, to mimic osteoporosis.
  • animals were fed a casein-based semi-synthetic diet with either the harvested hard deer antler and " deer bone test product, milk calcium, or calcium carbonate serving as the only source of calcium for the remaining 28 weeks of the study.
  • the sham control and OVX control group received base diet.
  • the 60 rats underwent in vivo dual energy X-ray absorptiometry (DEXA) scanning of bone density under anaesthesia. Blood samples were taken at week -1 , 4 and 32.
  • Figure 1 shows the size exclusion chromatography profiles of solutions of the extracts, each dissolved at 8 mg/ml in ammonium bicarbonate.
  • the overlaid plots for each sample measured are standardised to be on the same scale to allow direct comparison.
  • velvet as expected showed a strong peak.
  • the harvested hard deer antler also showed a similar strong peak activity to velvet.
  • the cast antler showed significantly less activity with only a small peak compared to that observed for velvet and harvested hard antler.
  • the ethanol washed sample showed minimal activity as well suggesting that this standard ethanol wash step removes potential bioactive.
  • This example describes an experiment completed by the inventors to further define the activity of hard antler powder fractions harvested from live deer on osteoclastogenesis and osteoblast proliferation compared to cast antler and velvet.
  • the purpose of the study was to test the effect of four antler extracts on bone osteoblast proliferation (increase in growth and number) or the formation of osteoclasts (bone resorbing cells).
  • the extract samples tested were:
  • Osteoclasts can be generated from the murine macrophage RAW 264.7 cell line.
  • RAW 264.7 cells were seeded into 24 well plates and treated with murine RANK-L.
  • RANK-L is an antibody that inhibits osteoclast cell function.
  • the effect of the hard antler powdered fractions on osteoclast formation was tested by the addition of the compound to the RANK-L containing cell culture media at different concentrations (0.1 , 1 , 10, 100 g/ml). The four samples to be test were solubilised in cell culture medium by sample weight.
  • solubilised samples were filter sterilised (0.2 ⁇ ) prior to being added to the cells.
  • Lactoferrin (1 15 ⁇ g/ml) was included as a positive control for its known anti- osteoclastogenic effects.
  • Each experiment (plate) was run in triplicate for replication. The cells were incubated for five days at 37°C in 5% C0 2 . The media and treatments were changed with fresh treatments on day 3. The plates were then fixed and stained for tartrate-resistant acid phosphatase (TRAP) and then counterstained with hematoxylin. Osteoclasts appear as large multinucleated cells staining purple red and may form even larger giant cells.
  • TRAP levels in the cell culture media of each well was measured colometrically at 550 nm, and expressed as treatment over the zero control.
  • C3T3-E1 preosteoblast cells (MC3T3-E1 subclone 4 murine preosteoblast, CRL-2593; ATCC, Manassas, VA, USA) were maintained under 5% C0 2 and 95% air at 37°C in MEMa medium (MEMa; Gibco, Invitrogen NZ) with 10% (v/v) FCS (Gibco, Invitrogen NZ) supplemented with antibiotics (Gentamicin; Gibco, Invitrogen NZ).
  • MEMa MEMa medium
  • FCS Gibco, Invitrogen NZ
  • the effect of the four test samples on osteoblast cell proliferation was determined by treating osteoblasts with different extract concentrations (0.1 , 1 , 10, 100, 1 ,000 pg/ml).
  • Cells were seeded in 96-well plates at 0.75x10 5 cells / ml (0.1 ml / well) and incubated for 24 hr at 37"C. After 24 hr, cell growth was arrested by incubation in medium with 0.1 % BSA for a further 24 hr. The fractions were dissolved in cell culture medium (w/w) and filter sterilised (0.20 ⁇ ). Lactoferrin is known to stimulate cell proliferation and was included as a positive control (1 ,000 g/ml). Each experiment (plate) was run in triplicate for replication. Cells were incubated with each concentration in medium with 0.1 % BSA. Each plate was incubated for 24 hr at the appropriate temperature and then cell proliferation was quantified via an MTT colorimetric assay.
  • the RAW cells are a mouse monocyte cell line that can be transformed into osteoclasts using RANK ligand.
  • the value for the 0 control is approximately 1 in Figure 2 and with lactoferrin (LF) is lower at 0.75. This means LF has suppressed the formation of osteoclasts.
  • the harvested hard antler fraction inhibited (Figure 3; p ⁇ 0.0001) osteoclast differentiation at a slightly wider range of concentration being 1 , 10 and 100 ⁇ g/ml. Large multinucleated osteoclasts were absent at 100 pg/ml. A 10 g/mL concentration of hard antler had a similar effect to lactoferrin. At 100 ⁇ g/mL hard antler was also more effective than lactoferrin.
  • Lactoferrin significantly increased the growth of the osteoblasts compared to the 0 control (MTT (treatment/control).
  • hard antler contain components that inhibit osteoclast cell development.
  • the cast antler and ethanol-washed harvested hard antler powder fractions do inhibit osteoclast development but, unlike the harvested hard deer antler, do not stimulate osteoblast cell proliferation.
  • the velvet powder fraction did not have any detectable anti-osteoclastic effect but exhibited an anabolic effect on the osteoblasts resulting in proliferation.
  • the purpose of the study was to test the effect of four antler extracts on bone osteoblast proliferation (increase in growth and number) or the formation of osteoclasts (bone resorbing cells).
  • the extracts tested were:
  • the cast antler and ethanol-washed hard antler powder fractions inhibited osteoclast development whilst not influencing osteoblast cell proliferation.
  • the harvested hard antler powder fraction had both an inhibitory effect on osteoclast development and also exhibited an anabolic effect on osteoblast cell proliferation.
  • the velvet powder fraction did not have any detectable effect on osteoclast development but had an anabolic effect on osteoblast cell proliferation.

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Abstract

L'invention concerne une formulation et ses procédés apparentés, ainsi que ses utilisations, cette invention utilisant le bois de cervidé dur qui est prélevé sur le cerf entre la période de dessèchement du bois et un mois avant son détachement. Ce bois de cervidé dur prélevé particulier s'avère présenter des effets inattendus, notamment une augmentation de la fonction cellulaire des ostéoblastes ou une diminution de la fonction cellulaire des ostéoclastes; ou une augmentation de la fonction cellulaire des ostéoblastes et une diminution de la fonction cellulaire des ostéoclastes.
PCT/NZ2010/000185 2009-09-15 2010-09-15 Composition active obtenue à partir de bois de cervidé et ses procédés d'utilisation WO2011034447A1 (fr)

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NZ577945 2009-09-15

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106880839A (zh) * 2015-12-14 2017-06-23 吉林省东鳌鹿业集团有限公司 一种鹿骨片及其制备方法
CN114306386A (zh) * 2022-01-04 2022-04-12 中国农业科学院特产研究所 鹿茸干细胞条件培养基的用途

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5408041A (en) * 1992-03-18 1995-04-18 Rhone-Poulenc Rorer Pharmaceuticals Inc. Process of purifying antler-derived bone growth factors

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5408041A (en) * 1992-03-18 1995-04-18 Rhone-Poulenc Rorer Pharmaceuticals Inc. Process of purifying antler-derived bone growth factors

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BENSKY D. ET AL: "Chinese Herbal medicine: Materia Medica, 3rd Edition", EASTLAND PRESS, SEATTLE, WA, USA, pages: 767 *
MENG HAI-YAN ET AL: "Effects of pilose antler and antler glue on osteoporosis of ovariectomized rats", ZHONG YAO CAI (JOURNAL OF CHINESE MEDICINAL MATERIALS), vol. 32, no. 2, 2009, pages 179 - 182 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106880839A (zh) * 2015-12-14 2017-06-23 吉林省东鳌鹿业集团有限公司 一种鹿骨片及其制备方法
CN114306386A (zh) * 2022-01-04 2022-04-12 中国农业科学院特产研究所 鹿茸干细胞条件培养基的用途

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