WO2013007807A1

WO2013007807A1 - Selected cimicifuga fractions for the treatment of osteoporosis

Info

Publication number: WO2013007807A1
Application number: PCT/EP2012/063748
Authority: WO
Inventors: Wolfgang Wuttke; Daniel INTELMANN; Markus Schuh; Guenther Stecher; Gudrun ABEL; Franz Jakob; Regina EBERT; Dana SEIDLOVÀ-WUTTKE
Original assignee: Bionorica Se
Priority date: 2011-07-12
Filing date: 2012-07-12
Publication date: 2013-01-17

Abstract

The invention relates to cimicifuga fractions for the prophylaxis and treatment of osteoporosis in humans and animals, to medicinal products thereof, and to methods for producing same.

Description

Selected Cimicifuga fractions for the treatment of osteoporosis

Besehreibung

The invention relates to Cimicifuga fractions for the prophylaxis and treatment of osteoporosis in humans and animals,

Drugs thereof and process for their preparation.

Cimicifuga extracts are already known for the treatment of diseases based on aqueous-ethanolic extracts. In particular, their dry extracts are used as phytopharmaceuticals.

In recent years, the use of the

Hormone replacement therapy (HRT) in climacteric post

menopausal complaints such as mood swings,

Hot flashes and increased sweating widespread. Increases in cardiovascular and thromboembolic disease, as well as increased breast cancer risk during HRT treatment indicate the search for a medication without the estrogen-related side effects. The Applicant therefore sells the Cimicifuga drug product Klimadynon® (dry extract from Cimicifuga rootstock) for the treatment of climacteric complaints.

Cimicifuga extracts are already described as estrogen-like organ-selective drug in WO1999047149A1.

WO200236140A discloses that preparations of Cimieifuga are therapeutically effective in the presence of urinary incontinence. Furthermore, WO1999047149A1 discloses the suitability of Cimicifuga extracts as an organ-selective drug for the treatment of osteoporosis and atherosclerosis.

However, there is a high interest in optimizing Cimicifuga extracts and providing improved (special) extracts.

Therefore, it is an object of the present invention to provide an improved pharmaceutical for the treatment of osteoporosis based on Cimicifuga.

Surprisingly, it could be found that certain Cimicifuga fractions for the treatment and prophylaxis of

Osteoporosis are particularly suitable.

In WO1999047149A1 is described that with organic

Solvents (dichloromethane) can be obtained by liquid-liquid extraction against water, an organo-selective extract from the organic phase, while the aqueous (residual) phase shows no activity (detection by a

Estradiol binding assay). Such an organo-selective

Extract is suitable for treatment of estrogen deficiency, including for the treatment of osteoporosis. On the basis of this prior art, fractions which show a bone-protective and anti-osteoporic action, both for the aqueous residual phase and for the organic phase, could surprisingly be obtained by means of a chromatographic step. In a preferred embodiment, the

chromatographic step based on a solid (stationary) phase, in particular by means of a column chromatography or phase chromatography, in particular a

Reverse phase chromatography, especially normal pressure silica-reversed phase chromatography, e.g. by means of a RP-C18 column. In a preferred embodiment, a

High performance liquid chromatography (HPLC) can be performed.

The chromatographic step allows the provision of fractions having a high content of active ingredients, which are also advantageously enriched, so that an effective lower dose level can be achieved over the prior art. In addition, the allowed

chromatographic step of the invention improved reproducibility and uniformity of the obtained

Fractions according to the invention.

The invention therefore relates to a process for the production of bone-protective and anti-osteoporic Cimicifuga fractions from a Cimicifuga extract, wherein at least one chromatographic step is carried out.

Furthermore, it is preferred that in a first step, a liquid-liquid extraction (LLE for short) of Cimicifuga takes place, wherein a first solvent is water or aqueous

Shares (such as a mixture of water and

Alcohol, 70:30 v / v, 50:50 v / v, 30:70 v / v) and the second

Solvent is an organic solvent. Organic solvents may preferably be such as Dichloromethane, ether, diethyl ether, tert. Butyl methyl ether (TBME), pentane, hexane, heptane. There are according to the invention an aqueous fraction ("C001 / R") and a lipophilic fraction

Preferably, the first solvent is water, and further the second solvent has such a preferred polarity as dichloromethane.

Therefore, the invention relates to a method for the production of bone-protective and anti-osteoporotic Cimicifuga fractions, wherein in a first step

i. ) a liquid-liquid extraction of Cimicifuga takes place and ii. ) the fractions obtained with at least one

separated chromatographic step and obtained in this way the bone-protective and anti-osteoporotic Cimicifuga fractions.

Furthermore, the invention relates to a process for the preparation of bone-protective and anti-osteoporotic Cimicifuga fractions, wherein in a first step

i. ) a liquid-liquid extraction of Cimicifuga with an aqueous phase takes place against an organic phase and ii. ) the fractions obtained with at least one

separated chromatographic step and obtained in this way the bone-protective and anti-osteoporotic Cimicifuga fractions. In another embodiment, the chromatographic step may be repeated. It is further preferred that the aqueous phase have high proportions of water and a pH of 7 to 9, preferably 8 to 8.5. If necessary, the pH value can be adjusted by means of a base (eg NaHCC> 3). In a further embodiment of the invention, the chromatographic step may preferably be carried out on the respective aqueous (C001 / R) or lipophilic (C001 / S) fraction.

There are obtained according to the invention fractions with

enriched with effective ingredients and consequently with smaller amounts an improved effect can be achieved. Advantageously, by means of the invention

Fractionation, such fractions are obtained, which achieve at the same dosage / amount / patient increased efficacy against C001 / R or C001 / S. Particularly advantageous such (sub) fractions (C001 / S1-3, see Table 1) can be obtained from the lipophilic fraction containing saponins, actin and / or deoxyactein, in particular, these ingredients are effective

Main components, or the (sub) fractions are with

Saponins, actein and / or deoxyactone enriched.

Particularly advantageous such (sub) fractions (C001 / R1-3, see Table 1) can be obtained from the aqueous fraction containing cimipronidine or Cyclocimipronidin, in particular, these ingredients are effective

Main components, or the (sub) fractions are with

Cimipronidine or cyclocimipronidine enriched. Furthermore, all (sub) fractions are preferred which contain cimipronidine or cyclocimipronidine and with

Cimipronidine or cyclocimipronidine are enriched, even if saponins, actin and / or deoxyactein are present.

For example, preferred fractions can be combined.

Further preferred embodiments of the fractions can be taken from Table 1, in particular by means of

set out ingredients for C001 / S1-S3 and C001 / R1-R3.

Surprisingly, fractions enriched with saponins, actin and / or deoxyactein, cimipronidine, cyclocimipronidine, are particularly advantageous

Bone-protective and anti-osteoporotic effect.

This is particularly surprising for the fractions from the aqueous phase of a liquid-liquid extraction, since in the prior art, the aqueous fraction have no saponins, but the inventive (sub) fractions from the aqueous phase can show a strong bone-protective and anti-osteoporotic effect ,

In addition, the inventive (sub) fractions from the aqueous phase show a beneficial effect against

Hot flushes, such as for peri- or post-menopausal complaints or climacteric complaints, are described.

Therefore, the invention also relates to a medicament containing Cimicifuga fractions for use in the

Treatment and prophylaxis of osteoporosis in humans and Animal, wherein at least one chromatographic step

is carried out.

The invention particularly relates to a pharmaceutical

containing Cimicifuga fractions for use in the

Treatment and prophylaxis of osteoporosis in humans and animals, wherein

i. ) a liquid-liquid extraction of Cimicifuga with an aqueous phase takes place against an organic phase and ii. ) the organic fraction (C001 / S) obtained is separated by at least one chromatographic step, and such fractions are obtained which contain saponins, actein and / or deoxyactein, if appropriate also petasiphenone,

Cimiracemate, cimiracemoside I, cimigenol; and / or ii. ) the aqueous fraction (C001 / R) obtained is separated by at least one chromatographic step, and such fractions are obtained which contain cimipronidine or

Cyclocimipronidine, if necessary, continue to contain caffeic acid, methylserotonin, (iso) ferulic acid, fulkiic acid, petasiphenone, petasiphenol, cimiciphenone, Cimiracemat, cimicifugic acid, fukinolic acid; iii. ) optionally the fractions obtained from ii.) are combined.

In the context of this invention, the term "Cimicifuga" (black cohosh) is originally from North America

originating (medicinal) plant Cimicifuga racemosa (CR) - too Native American female root - understood, this is about 2 m tall and has silvery-white flowers that form long clusters. The dried rootstock is used medicinally with the roots (drug). In accordance with the invention

also the following species: C. foetida, C. dahurica, C. heracleifolia, C. simplex, C. japonica, C. europaea, C.

acerina, C. elata, C. rubifolia. Furthermore, rhizomes, roots, stems, leaves and / or petals of the plants can be used. The skilled person is able to produce from Cimicifuga plant parts, in particular root, suitable "Cimicifuga extracts" according to claim 1, as already commercially available, eg Klimadynon® (supra.) In the literature, a suitable (aqueous-ethanolic) extract as "CR BNO 1055 ". For example, For example, extracts or fractions according to the invention can be prepared by means of organic solvents, such as alcohols, in particular

Ethanol, dichloromethane, heptane and the like. to be obtained. Furthermore, aqueous extracts or fractions are possible. Also included according to the invention and preferred are corresponding

Dry extracts, e.g. the well-known "BNO 1055".

Described ingredients of Cimicifuga or Cimicifuga extracts may be: triterpenes such as acetol and

Cimicigenolderivate, polyphenols, such as caffeic acid and its derivatives, chlorogenic acid, piscidic acid, fucinoleic acid, ferulic and isoferulic acid, Cimicifuginsäuren, formononetin, alkaloids

U. · cL · ·

The Cimicifuga fractions can be prepared by the method according to the invention. Therefore, the invention relates to Cimicifuga fractions obtainable by a process according to the invention or the Cimicifuga fractions are obtained by a process according to the invention. Therefore, the invention relates to a process for the preparation of a medicament (pharmaceutical) or pharmaceutical

Composition or use of a drug

(Medicament) or pharmaceutical composition containing Cimicifuga fractions, in particular for use in the treatment and prophylaxis of osteoporosis in humans and animals, if necessary. Containing other auxiliaries and additives.

In the context of this invention is under "osteoporosis" the

Disease of the skeletal system with loss or reduction of bone structure and structure and increased

Fracture susceptibility understood. For further definition see e.g. Pschyrembel, de Gruyter, 261st edition, Berlin 2007.

Therefore, the invention also relates to a medicament according to the invention for the treatment and prophylaxis of osteoporosis containing Cimicifuga fractions for the treatment and prophylaxis of osteoporosis in humans and animals, if necessary. Other auxiliaries and additives.

Furthermore, the invention therefore relates to a pharmaceutical

Formulation containing a medicament according to the invention (also called "active ingredient" or "composition"). Examples of particularly suitable pharmacologically acceptable carriers are known to those skilled in the art and include buffered Saline solutions, water, emulsions such as oil / water emulsions, various types of detergents, sterile

Solutions, etc.

Pharmaceutical compositions comprising such carriers can be formulated by known conventional methods. These pharmaceutical compositions can be administered to an individual / patient in a suitable dose. Administration can be oral, parenteral, intravenous, intraperitoneal, subcutaneous, intramuscular, local, intranasal, or intradermal, or via a catheter at one site in an artery. The type of dosage is from

attending physician according to the clinical factors

certainly .

In the context of this invention is the oral and subcutaneous

Application preferred.

It is known to the person skilled in the art that the type of dosage depends on various factors, e.g. the body size or the weight, the body surface, the age, the

Gender or the general health of the patient, but also on the specific agent to be administered, the duration and route of administration, and other medications that may be administered in parallel.

The medicaments according to the invention can be in the form of

pharmaceutical preparations in dosage units

be prepared. This means that the preparation is in the form of individual parts, for example tablets, dragees, capsules, pills, suppositories and ampoules, whose active substance content a fraction or a multiple of a single dose. The dosage units may be, for example, 1, 2, 3 or 4 single doses or 1/2, 1/3 or 1/4 of a single dose

contain. A single dose preferably contains the amount of active ingredient which is administered in one application and which usually corresponds to a whole, a half, a third or a quarter of a daily dose.

Among non-toxic, inert pharmaceutically acceptable

Carriers are solid, semi-solid or liquid

To understand diluents, fillers and formulation aids of any kind.

Preferred pharmaceutical formulations are tablets, dragees, capsules, pills, granules, suppositories, solutions, juice, suspensions and emulsions, pastes, ointments, gels,

Creams, lotions, powders and (nose) called sprays. Tablets, dragees, capsules, pills and granules may contain the active ingredient (s) in addition to the usual excipients, such as a) fillers and extenders, e.g. Starches, lactose, cane sugar, glucose, mannitol and silicic acid, b) binders, e.g.

Carboxymethylcellulose, alginates, gelatin,

Polyvinylpyrrolidone, c) humectants, e.g. Glycerine, d) disintegrants, e.g. Agar-agar, calcium carbonate and

Sodium carbonate, e) dissolution inhibitor, e.g. Paraffin and f) absorption enhancer, e.g. quaternary ammonium compounds, g) wetting agents, e.g. Cetyl alcohol, glycerol monostearate, h) adsorbents, e.g. Kaolin and bentonite and i)

Lubricants, for example talc, calcium and magnesium stearate and solid polyethylene glycols or mixtures of the substances listed under a) to i). The tablets, dragees, capsules, pills and granules can be mixed with the usual, optionally opacifying

and may also be composed in such a way that they give off the active substance only or preferably delayed in a certain part of the intestinal tract, optionally delayed, as embedding masses e.g. Polymer substances and waxes can be used.

Furthermore, it is preferred that the galenic form a

Coated tablet, in particular a calcium coated tablet, as disclosed in WO2002100422.

The active ingredient (s) may optionally also be incorporated with one or more of the excipients listed above

microencapsulated form.

Suppositories may contain, besides the active ingredient (s), the usual water-soluble or water-insoluble excipients, e.g. Polyethylene glycols, fats, e.g. Cocoa fat and higher esters (e.g., C14 alcohol with C16 fatty acid) or

Mixtures of these substances.

Ointments, pastes, creams and gels may contain, in addition to the active substance (s), the usual excipients, e.g.

animal and vegetable fats, waxes, paraffins, starch, tragacanth cellulosic derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide or mixtures of these substances.

Powders and sprays may contain, in addition to the active substance (s), the usual excipients, for example lactose, talc, Silica, aluminum hydroxide, calcium silicate and

Polyamide powder or mixtures of these substances. Sprays may additionally contain the usual propellants.

Solutions and emulsions may contain, in addition to the active substance (s), the customary carriers such as solvents, solubilizers and emulsifiers, e.g. Water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, in particular cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil, glycerol, glycerol formal,

Tetrahydrofurfuryl alcohol, polyethylene glycols and

Fatty acid esters of sorbitan or mixtures of these substances.

Suspensions may in addition to the active substance or the

conventional carriers such as liquid diluents, e.g.

Water, ethyl alcohol, propylene glycol, suspending agent, e.g. ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose,

Aluminum metahydroxide, bentonite, agar-agar and tragacanth or mixtures of these substances. The mentioned

Formulations may also include colorants,

Preservatives and odor and flavor additives, such as peppermint oil and eucalyptus oil and sweeteners, such as saccharin included. For the preparation of the Cimicifuga extracts according to the invention and moreover reference is made to the technical teachings which are the subject of EP 1368605B1, EP 0753306B1. The following examples and figures serve to illustrate the invention, without the invention to these examples

restrict .

Examples: Example 1

The Cimicifuga racemosa dry extract (C001) was over

Dichloromethane-water (pH 8.5) worked up in two fractions. The water fraction (C001 / R) contains both the simple phenylpropanoids and the cimicifugic acids, while in the dichloromethane phase (C001 / S) the saponins are enriched.

Starting from the dry extract, 65% of the

Ingredients in the water-soluble phase and 20% of

Ingredients in the lipophilic phase again. The 100% missing 15% of the ingredients are in the mixed phase (does not correspond to the boundary layer between the two

Miscible liquids during the liquid-liquid

Work-up), which was analyzed analytically but not further worked up. These 15% correspond in theirs

Composition of the whole extract.

The thin - layer chromatographic examination of the

prepared fractions is shown in FIG. In the water-soluble fraction, both simple (Rf at 0.7-0.8) and compound phenylpropanoids (Rf at 0.2-0.3) are present (control by thin-layer chromatography). Furthermore, the presence of methylserotonin could be confirmed by mass spectroscopic analysis.

In the lipophilic fraction saponins are found again. This was confirmed both by thin layer chromatography and by HPLC-MS.

FIGURE 1 also shows an analytical comparison between different Cimicifuga extracts prepared at different times (lanes 1-5).

The fractions (LLE fraction) shown in FIGURE 1, namely water soluble fraction (= residual fraction, C001 / R) and lipophilic fraction (= saponin fraction, C001 / S) were subjected to further subfractionation by means of a chromatographic step and the subfractions were assayed for their bone protection and anti-osteoporotic effects. The subfractionation is carried out by means of a workup of the LLE fractions over normal pressure silica

Reverse phase chromatography. The obtained subfractions were analyzed analytically and finally combined into three characteristic pools (C001 / R1, C001 / R2, C001 / R3 and C001 / S1, C001 / S2, C001 / S3).

Recovery of Subfractions from Saponin and Residual Fraction by Column Chromatography

The two fractions (C001S, C001R) become 3 each

Subfractions further processed. For this purpose, the fractions are applied to a silica gel RP-C18 packed glass column and then eluted with increasing methanol concentration. About a fraction collector are 20 ml aliquots

collected, which were examined by thin layer chromatography. Based on the results of

Thin layer chromatography, the aliquots were pooled. After concentrating the pools on Rotavapor they are frozen and dried on the lyophilizer, whereby the sub-fractions were recovered. The fractionation protocol looks in detail like this:

The saponin fraction is dissolved in methanol (1 ml per gram

Saponin fraction) dissolved, then added the same amount of water and the resulting suspension to the column

given up. Elution is successively with 50%, 60%, 80% and 100% methanol. The 3 subfractions Sl (26% of the amount of saponin fraction used), S2 (37%), and S3 (37%) are obtained.

The residual fraction is suspended in 10% methanol (1.25 ml per gram of residual fraction) and applied to the column in this form. Elution is carried out successively with 10%, 30% and 70% methanol. After combining the corresponding aliquots, the 3 subfractions R1 (72% of the residual fraction used), R2 (19%), and R3 (9%) are present.

Appropriate controls were performed by thin layer chromatography.

Table 1 Ingredients in the Cimicifuga fractions

Ingredient Mass C001 C001 C001 C001 C001 C001

/ Sl / s2 / S3 / Rl / R2 / R3 γ-guanidino-129, 1 +

butyraldehyde 603

Cyclocimipronidine 153, 1 ++ ++ +++

817

Cimipronidine 171.1 ++ ++ +++

007

Salsolinol 179.2 +

157

Fructose 180, 1 +++ +++

559

Glucose 180, 1 +++ +++

559

Inositol 180, 1 +++ +++

559

Caffeic acid 180, 1 +++ +++

574

Νω-methyl serotonin 190.2 +++

416

Ferulic acid 194, 1 ++ ++

840 I soferulic acid 194, 1 +++

840

Piscidic acid 256.2 ++ ++

088

Fukicin 272.2 ++

082

Petasiphenon 330, 2 ++ ++

888

Sucrose 342.2 +++

965

Cimiciphenone 344.3 ++

154

Petasiphenol 344.3 ++

154

Cimiciphenol 358.3 ++ ++

Cimiracemate A 420

Cimiracate B 358.3 ++ ++

420

Cimiracate C 388.3 ++ ++

680 Cimiracate D 388.3 ++ ++

680

Cimicifugic acid D 418.3 ++

509

Cimicifugic acid E 432, 3 ++

775

Cimicifugic acid F 432, 3 ++

775

Fukolinic acid 434, 3 +

503

Cimicifugic Acid A 448.3 ++

769

Cimicifugic acid B 448.3 ++

769

Raffinose 504, 4 +++

371

Cimiracemoside I 600.3 ++ ++ ++

662

Cimiaceroside A 618.3 ++

767 Cimicifugoside M / 620, 3 +++ cimiracemoside C 924

Cimigenol 620, 3 +++ xylopyranoside 924

25-0- 634, 8 +++

Methylcimigenol-3-403

O-arabinoside

25-0- 634, 8 ++

Methylcimigenol-3-403

O-xylopyranoside

26-658.3 +++

Deoxycimicifugoside 716

23-epi-26-660, 3 +++ +++

Deoxyactein 873

Cimiracemoside J 660.3 ++

873

Cimiracemoside K 660.3 ++

873

Cimiracemoside N 660.3 ++

873 23-0- 662, 4 +++ +++

Acetylshengmanol 029

3-0-beta-D-xylopyranoside

25-0- 662, 8 +++ +++

Acetylcimigenol 504

arabinopyranoside

25-0- 662, 8 +++ +++

Acetylcimigenolxyl 504

opyranosid

Actein 676, 3 +++ +++

822

Cimiracemoside F 676, 3 +++ +++

822

Cimiracemoside G 676, 3 ++ ++

822

Cimiracemoside L 704, 4 ++

135

Cimiracemoside M 704, 4 ++

135 STRUTS analysis of Cimicifuga fractions (esp. C001 / R1, C001 / R2 and C001 / R3 and C001 / S1, C001 / S2 and C001 / S3):

The Struts analysis determines the connectivity of the trabecular bone. The ratio between the trabeculae points (NODES) and the trabecular ends (STRUTS) serves as a measure. The more Nodi and the less free trabecular ends can be determined, the more stable is the Trachea Net.

This method is helpful in order to be able to make a more specific statement about the connectivity of cancellous bone. The trabecular structure becomes node points (NODES) and

Strokes (STRUTS) simplified. A node is defined as a point of association of three or more trabeculae.

A free end is defined as a point at which no

Clash of trabeculae takes place. Struts, as the individual trabeculae, are expressed as percent of the total

Length expressed.

Node Strut Analysis Parameters:

Number of Trabecular Nodes (No.Nodes [# /])

· Number of free trabecular ends [# / mm2]

• Total strut length (TSL [mm / mm2])

Strut length from free trabecular ends to free trabecular ends (SL.FF [% of TSL])

• Strut length of trabecular node points too

Trabecular node points (SL.NN [% of TSL])

Design : ■ 10 test animal groups of 10 animals

^■ oral administration of:

a) the respective weight equivalents of

Fractions / subfractions (C001 / S, C001 / S / 1-3, C001 / R and C001 / R1-3) with respect to C001 total extract (10 mg

Total extract / animal)

b) Negative control: phytoestrogen poor feed ("Ko") c) Positive control: estradiol benzoate 10 mg / kg animal ("E2")

Treatment duration: 4 weeks

Main parameters: weight gain, feed intake,

bone density

Results: a. ) It could be shown that the saponin fraction

(C001 / S / 1-3) bone protection (FIG. 3) and the

Residual fractions (C001 / R / 1-3) have a bone-protective effect (FIG. 4),

b. ) The fraction C001 / S / 2 has the strongest

Bone-protective activity of the three saponin subfractions (FIG. 3),

c. ) The third of the 3 sub-fractions of the residual fraction

(C001 / R / 3) shows strong bone-protective activity (FIGURE 4).

A Quantitative Computed Tomography (qCT) enables rapid and accurate determination of rat bone mineral density. The first in cancellous bone of the

Limb bones of ovariectomized rats beginning Bone loss can be clearly detected with qCT as early as four weeks after ovariectomy.

As a result, the potential bone-protective and anti-osteoporic effect of the fractions from a Cimicifuga extract can be demonstrated.

By determining the subcutaneous temperature during the rat experiment described above after treatment with COOI / S, C001 / S / 1-3 or C001 / R and C001 / R / 1-3, it can be confirmed that the residual fraction and especially the subfraction C001 / R and C001 / R1-3 and not the saponin fraction

Subfractions advantageous the "hot-flushes" can reduce (FIGURE 5).

Example 2:

Studies on the adipogenic differentiation potential of C001, COOl / S and C001 / R in hMSCs (human mesenchymal

stem Cells)

To complete, to those with the proportionately stimulated stem cells with plant extract, were more

Differentiation studies (n = 3) each with 0, 1 ng / ml, 10 ng / ml, 100 ng / ml and 1000 ng / ml of said extracts

carried out .

200,000 cells / well were seeded on 6-well plates and adipogenously differentiated after reaching confluence with 1, 10, 100 and 1000 ng / ml with C001, C001 / S and C001 / R for 3 weeks (DMEM (hG); % Penicillin / streptomycin; 10% fetal Calf serum; 0.1% indomethacin, dexamethasone and IBMX; 0.01% insulin). Subsequently, the cultures were stained with oil red. The quantitative evaluation was carried out with the

Evaluation software Axio Visio from Zeiss. An adipogenic

Control was included (see FIG. 6).

In all three extracts tested there is a decrease in adipogenic differentiation in the examined

Concentrations. Only at 10 ng / ml C001 / S and 1000 ng / ml C001 / R is no influence measurable. Example 3:

Studies on the osteogenic differentiation potential of C001, C001 / S and C001 / R in SAOS-2 cells (preosteoblastic osteosarcoma cell line)

200,000 cells / well were seeded on 6-well plates and after reaching confluence with 1, 10, 100 and 1000 ng / ml with

C001, C001 / S and C001 / R for 2 and 4 weeks osteogenic

differentiated (n = 3) (DMEM (hG); 1% penicillin / streptomycin;

10% fetal calf serum; 1% β-glycerol phosphate; 0.01%

Dexamethasone). Subsequently, the staining of the

Cultures with alizarin red-S. The quantitative evaluation was carried out with the Axio Visio evaluation software from Zeiss.

An osteogenic control was included (see FIGURE 7).

In the osteogenic differentiation of SAOS-2 with C001 an increase of about 10% with stimulation at 10 ng / ml is observed after 4 weeks. This effect is not measurable after 2 weeks. After stimulation with C001 / S increases Differentiation capacity after 2 weeks up to 20% in the investigated concentrations. After 4 weeks no

Difference more measurable. In the water-soluble fraction C001 / R, an increase in osteogenic differentiation in the lower concentrations is quantifiable, this effect decreases as the concentration increases.

Example 4:

Transdifferentiation studies of 100 ng / ml and 1000 ng / ml C001 / S in hMSCs

For this purpose, the cells to be stimulated were seeded in 6-well plates at a concentration of 100,000 cells / well and cultured until reaching confluence (80%) at 37 ° C. Then there was a two-week differentiation to adipocytes with and without extract and then a four-week

Differentiation to osteocytes (n = 3). This was followed by staining of the cultures with Alizarinrot-S. The data was evaluated with AxioVision rel 4.6 software from Zeiss.

Transdifferentiation capacity in favor of osteogenic differentiation increases at all concentrations tested, regardless of the time of stimulation (see FIGURE 8).

Statistics used:

Mann-Whitney U test: P <0.05 *; P <0.01 **; P <0.001 *** Conclusion from Examples 2 to 4: The plant extract from Cimicifuga racemosa and the investigated fractionated extracts C001 / S and C001 / R definitely show potential to increase osteogenic activity

Mineralization and reduction of adipogenic

Differentiation. Since osteoporosis often occurs in old age and is accompanied by an increase in bone marrow fatting, the results show that Cimicifuga fractions

Have potential for the prevention and treatment of osteoporosis. Furthermore show the

Transdifferentiation studies that in particular the extract C001 / S after a two-week adipogenic differentiation is capable of the differentiation potential in the direction

To increase osteocytes by about 15%.

Explanations to the figures: Figure 1: thin layer chromatography different

Cimicifuga racemosa dry extract lots and manufactured fractions. 1 = C002, 2 = C004, 3 = C003, 4 = C001, 5 = lot 97042148, 6.7 = water fraction C001, 8.9 = lipophilic fraction C002. 10 = reference standard isoferulic acid (phenylpropanoid), 11 = reference standard deoxyactein (saponin).

Above: UV detection, colors inverted; Rf 0.2 - 0.3

Cimicifugic acids, Rf 0.7-0.8 simple phenylpropanoids.

Bottom: detection after reaction with anisaldehyde / sulfuric acid. Red colored bands correspond to saponins. Figure 2 gives an overview of the fractionation carried out and to the main components, which in the individual

Subfractions were detected. Legend: "Saponin fraction (= C001 / S)" is the dichloromethane fraction (supra) and "R" Fraction "is the aqueous fraction (C001 / R)," TE "= dry extract (starting extract = C001). The (sub-) fractions C001 / S1, C001 / S2 and C001 / S3 are determined by means of a

chromatographic step from C001 / S obtained (supra). The (sub-) fractions C001 / R1, C001 / R2 and C001 / R3 are obtained by means of a chromatographic step of C001 / R

(supra).

FIG. 3 shows the spongosia density measured by qCT in the metaphysis of the tibia with respect to the fractions and subtractions according to FIG. Legend after Figure 2.

FIG. 4 shows the spongosia density measured by qCT in the metaphysis of the tibia with respect to the fractions and subtractions according to FIG. Legend after Figure 2.

Figure 5 shows the effect of the subcutaneous temperature on the ovx rats to the fractions and subfractions according to. Legend after Figure 2.

Figure 6: Studies on the adipogenic

Differentiation potential of C001, C001 / S and C001 / R in hMSCs

Figure 7: Studies on the osteogenic

Differentiation potential of C001, C001 / S and C001 / R in SAOS-2 cells

Figure 8: Transdifferentiation studies of 100 ng / ml and 1000 ng / ml C001 / S in hMSCs

Claims

claims

A process for the preparation of bone-protective and anti-osteoporic Cimicifuga fractions from a Cimicifuga extract, wherein at least one chromatographic step is performed.

A process for the preparation of bone-protective and anti-osteoporotic Cimicifuga fractions according to claim

1, wherein in a first step i.) A liquid-liquid extraction of Cimicifuga takes place and ii.) The fractions obtained are separated with at least one chromatographic step and the fractions are obtained.

2, wherein in a first step i.) A liquid-liquid extraction of Cimicifuga with an aqueous phase takes place against an organic phase.

3, wherein the aqueous phase has high proportions of water and / or the organic phase from the group of dichloromethane, ether, diethyl ether, tert. Butyl methyl ether (TBME), pentane, hexane or heptane is selected.

A process for the preparation of bone-protective and anti-osteoporotic Cimicifuga fractions according to claim 3, wherein the aqueous phase has a pH of 7 to 9, preferably 8 to 8.5.

Process for the preparation of bone protective and Anti-osteoporotic Cimicifuga fractions according to any one of the preceding claims, wherein the chromatographic step by means of a solid phase, in particular by means of column chromatography,

Phase chromatography, reverse phase chromatography, normal pressure silica reverse phase chromatography, or high performance liquid chromatography (HPLC).

Cimicifuga fraction obtainable according to one of the preceding claims, optionally dried and comminuted.

Cimicifuga fraction obtainable according to one of the preceding claims, characterized in that

Saponins, Actein and / or Deoxyactein are or are enriched and / or Cimipronidin or Cyclocimipronidin contained or enriched.

Pharmaceutical composition containing a

Cimicifuga fraction according to one of the preceding claims, in the form of powder, granules, tablets, dragees, in particular a coated tablet or calcium coated tablet or in a galenic formulation, in particular an oral formulation such as dragees, tablets, film-coated tablets, capsules, liquid dilutions, in particular drops , Juices, syrups or a formulation for topical use such as sprays, ointments, emulsions, powders, powders, liquid or solid preparations for inhalation, compresses or lyophilisate, together with suitable additives and auxiliaries.

Medicament containing a Cimicifuga fraction according to one of the preceding claims for use in the Treatment and prophylaxis of osteoporosis in humans and animals.

11. Medicaments containing Cimicifuga fractions for use in the treatment and prophylaxis of

Human and animal osteoporosis, i.) Liquid-liquid extraction of Cimicifuga with an aqueous phase against an organic phase; and ii) separation of the obtained organic fraction with at least one chromatographic step, and obtaining such fractions which Saponins, Actein and / or Deoxyactein contain, if necessary. Continue

Petasiphenone, cimiracate, cimiracemoside I, Cimigenol. and / or ü.) the aqueous fraction obtained is separated by at least one chromatographic step, and such fractions are obtained which contain cimipronidine or cyclocimipronidine, if appropriate. furthermore caffeic acid, methylserotonin, (iso) ferulic acid, fulkiic acid,

Petasiphenone, Petasiphenol, Cimiciphenone, Cimiracemat,

Cimicifugic acid, fucinolic acid. iii.) if appropriate, the fractions obtained from ii.) are combined.