GB2534228A - Compound or mixture of compounds for the treatment of neurodegenerative diseases or oxidative stress injuries - Google Patents

Abstract

A composition for the treatment and prevention of neurodegenerative disease and injuries induced by oxidative stress comprising coptisine or an extract from Coptis rhizome including pharmaceutically acceptable salts, tautomers and stereoisomers of the compound, including mixtures thereof in all ratios, is provided. Preferably the coptisine is combined with at least one further compound selected from the group including berberine, jatrorrhizine and palmitine including pharmaceutically acceptable salts, tautomers and stereoisomers of the compounds, including mixtures thereof in all ratios. The neurodegenerative disease may be selected from the group including Alzheimers disease and other dementias, brain cancer, degenerative nerve disease, encephaltitis, epilepsy, genetic brain disorders, head and brain malformations, hydrocephalus, stroke, Parkinsons disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS or Lou Gehrigs disease) Huntingtons disease and prion diseases. Coptisine or an extract from Coptis rhizome for modulating the activity of thioredoxine-interacting protein is also provided.

Description

Intellectual Property Office Application No. GII1500867.5 RTM Date:2 October 2015 The following terms are registered trade marks and should be read as such wherever they occur in this document: Li_ahtcycler Mitotracker RNeasy

SYBR

Axioskop Intellectual Property Office is an operating name of the Patent Office www.gov.uk /ipo Title: Compound or mixture of compounds for the treatment of neurodegenerative diseases or oxidative stress injuries

Description

Field of the invention

[0001] The present disclosure provides a compound or a mixture of compounds for the treatment of neurodegenerative diseases or injuries associated with oxidative stress.

Background of the invention

[0002] In recent years many studies focused on cellular injuries related to oxidative stress and natural antioxidants with neuroprotective potential. A plurality of the investigated natural herbal extracts or single compounds exerts their protective effect via removal of surplus reactive oxygen species (ROS) or by the prevention of ROS generation. However, to enhance the protective effect of those antioxidants, other natural pharmaceuticals, which act via a different mechanism, are needed to achieve an additive or even synergistic effect. The concept of combining different natural products for the treatment of a certain disease was known, used and further improved in Chinese medicine for more than thousand years.

[0003] The dried root of Coptis chinensis Franch. (CR), also known as Chinese gold-thread (huang-lian in Chinese) has been used alone or in combination with other herbs as a traditional Chinese herbal medicine for more than a thousand years. In ancient times it was commonly used for inflammatory disease, dysentery or diabetes mellitus. Recent studies reported efficiency of CR on neurodegeneration (Luo et al., 2011; Han et al., 2012), apop-tosis (Miura et al., 1997; Friedemann et al., 2014), cancer (Tan et al., 2014; Zhu et al., 2014) and inflammation (Marinova et al., 2000). Furthermore, there is rising evidence that crude extract of CR is effective for the treatment of neurodegenerative disease associated with oxidative stress (Jung et al, 2009; Lo et al, 2012; Gong et al., 2012; Friedemann et al., 2014). However, until now the main active ingredients for the treatment of neuro-degenerative disease associated with oxidative stress are unknown.

[0004] Reactive oxygen species (ROS) have a significant impact on the development of neurodegenerative disease like Alzheimer or Parkinson's disease (Smith et al., 1996; Finkel et al., 2000; Perry et al., 2002; Vincent et al., 2004; Perfeito et al., 2012). Usually cells, including neurons, are well protected from ROS induced cytotoxicity by the endogenous antioxidant system. However, if the oxidative stress exceeds the antioxidative capacity of this system it can lead to DNA demethylation, histone acetylation, oxidative protein and lipid modification, increase of intracellular Cat-ions, depolarization of the mitochon-drial membrane potential (MMP), release of cytochrome c into the cytosol and as a consequence to apoptosis or necrosis (Gorman et al., 1996; Smith et al., 1996; Vincent et al., 2004; Rush et al., 1986; Coleman et al, 1989; Guidarelli et al., 1997; Amoroso et al., 1999; Martin et al, 2001; Nieminen et al, 1995, 1997; Zhao et at 2005; Kowaltowski et al, 2001; Liu et al., 1996; Li et al., 1997). For this reason, therapeutic strategies targeting ROS induced cytotoxicity are needed and could have a major impact on the treatment of neurodegenerative diseases which are associated with oxidative stress.

[0005] Recently it was possible to show that treatment of SH-SYSY neuroblastoma cells with tert-butylhydroperoxide (t-BOOH) induced intracellular ROS generation, leads to reduction of cell viability, decrease of the MMP and apoptosis (Friedemann et al., 2014). However, 24 h pretreatment with the watery extract of CR significantly attenuated all those effects, except of the ROS production. The results revealed furthermore that CR mediated down-regulation of TXNIP (an inhibitor of thioredoxin) might be essential for the cytopro-tective effect.

[0006] It is an object of the present disclosure to provide the main compound of CR, which is responsible for the cytoprotective effect of the CRE.

Summary of the invention

[0007] The present disclosure provides a composition for the treatment and prevention of neurodegenerative diseases and injuries induced by oxidative stress comprising coptisine or an extract from Coptis rhizome including pharmaceutically applicable salts, tautomers and stereoisomers of the compound, including mixtures thereof in all ratios.

[0008] Coptisine as a composition of the present disclosure may be combined with at least one further compound selected from the group comprising berberine, jatrorrhizine and palmatine including pharmaceutically applicable salts, tautomers and stereoisomers of the compounds, including mixtures thereof in all ratios.

[0009] The neurodegenerative diseases shall be selected from the group comprising Alzheimer's Disease and other dementias, Brain Cancer, Degenerative Nerve Diseases, Encephalitis Epilepsy, Genetic Brain Disorders, Head and Brain Malformations, Hydrocephalus, Stroke, Parkinson's Disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease), Huntington's Disease and Prion Diseases.

[0010] The oxidative stress induced injury may be selected from the group comprising cancer, Parkinson's disease, Alzheimer's disease, atherosclerosis, heart failure, myocardial infarction, fragile X syndrome, Sickle Cell Disease, lichen planus, vitiligo, autism, infec-tion, chronic fatigue syndrome and diabetes.

[0011] Another object of the present disclosure is a method for the use of coptisine or an extract from Coptis rhizome for the treatment of neurodegenerative diseases or oxidative stress induced injuries.

[0012] The coptisine may be combined with at least one further compound selected from the group comprising berberine, jatrorrhizine and palmatine including pharmaceutically applicable salts, tautomers and stereoisomers of the compounds, including mixtures thereof in all ratios [0013] The neurodegenerative disease may be selected from the group comprising Alzheimer's Disease and other dementias, Brain Cancer, Degenerative Nerve Diseases, Encephalitis Epilepsy, Genetic Brain Disorders, Head and Brain Malformations, Hydrocephalus, Stroke, Parkinson's Disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease), Huntington's Disease, Prion Diseases.

[0014] The neurodegenerative disease may specifically be Parkinson's disease.

[0015] The oxidative stress in a method for using coptisine or an extract from Coptis rhi-zome for the treatment of neurodegenerative diseases or oxidative stress induced injuries may comprise an induced injury being selected from the group comprising cancer, Parkinson's disease, Alzheimer's disease, atherosclerosis, heart failure, myocardial infarction, fragile X syndrome, Sickle Cell Disease, lichen planus, vitiligo, autism, infection, chronic fatigue syndrome and diabetes.

[0016] Another object of the present disclosure is method for the use of coptisine or an extract from Coptis rhizome for modulating the activity of thioredoxine interacting-protein.

Brief description of the Figures

[0017] Examples and embodiments of the present disclosure will be described and shown in the figures. It is obvious for a person ordinary skilled in the art, that the present invention is not limited to the shown embodiments. It shows: [0018] Figure 1 High-performance liquid chromatogram of CRE and mixed standard compounds (MSC).

[0019] Figure 2 DPPH-scavenging activity of Ber, Cop, Jat, Pal, NAC and Trolox.

[0020] Figure 3 Trolox equivalent antioxidant capacity (1EAC) of Ber, Cop, Jat and Pal [0021] Figure 4 Effect of CR main alkaloids on cell viability in SH-SY5Y cells [0022] Figure 5 Effect of CRE and Cop on cell viability in SH-SY5Y cells [0023] Figure 6 Effect of CRE and Cop on ROS level in SH-SY5Y cells [0024] Figure 7 Effect of 10004 CRE or 20gM Cop on t-BOOH-induced apopto-sis [0025] Figure 8 Effect of CRE and Cop on MMP [0026] Figure 9 Relative gene expression ratio (rER) of TXN1P [0027] Figure 10 TXNIP protein concentration of 1mg extracted total protein [0028] Figure 11 Neuroprotective effect of CRE and its main alkaloids on MPP+ induced neurotoxicity in SH-SY5Y cells Effect of 100 1.tIVI CRE on MPP+ induced apoptosis Effect of CRE on MPTP induced movement disorder Effect of CRE on movement disorders measured by the pole test Effect of CRE on TH+ cells in the substantia nigra and ventral tegmental area [0029] Figure 12 [0030] Figure 13 [0031] Figure 14 [0032] Figure 15

Detailed Description of the figures

[0033] The following abbreviations are used to describe the invention: AAPH 2,2"-Azobis(2-methylpropionamidine) dihydrochlorid ALS Amyotrophic Lateral Sclerosis AMP Adenosin-5'-monophosphat ASK Apoptosis signal-regulating k nase Ber Berberine Cat Calcium ion Cop Copti sine CR Coptis chinensis Franch CRE Coptis chinensis Franch watery extract DANN Deoxyribonucleic acid DAT Dopamine transporters DCF 2',7'-Dichlorofluorescein DDW Distilled de-ionized water DM SO Di m ethyl sulfoxid DPBS Dulbecco's phosphate buffered saline DPPH 2,2-Di(4-tert-octylpheny1)-1-picrylhydrazyl H2DCFH-DA 2',7"-Dichlorodihydrofluorescein diacetate HPLC High performance liquid chromatography i.p. Intraperitoneal Jat Jatrorrhizine MIMP Mitochondrial membrane potential MPP+ 1-Methyl-4-phenylpyri di ni um MPT Mitochondrial Permeability Transition MPTP 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine MSC Mixed standard compounds MTND1 Mitochondrially encoded NADH dehydrogenase 1 MTT Thiazolvl blue tetrazolium bromide No Number ORAC Oxygen Radical Absorbance Capacity Pal Palmatine PD Parkinson's disease QRT-PCR Quantitatv real-time polymerase chain reaction rER Relative gene expression ratio ROS Reactive oxygen species SEM Standard error of the mean SHXT San-Huang-Xie-Xin-Tang SN Substantia nigra SNpc Substantia nigra pars compacta SPB Sodium phosphate buffer SRCMO Shanghai Research Center For Model Organisms t-BOOH Tert-butylhydroperoxide TEAC Trolox equivalent antioxidant capacity TH+ Tyrosine hydroxylase positive Trx Thioredoxin t-sum Total time required to climb down t-turn Time the mice needed to turn TXNIP Thioredoxin-interacting protein VTA Ventral tegmental area [0034] Watery extraction of CR yields 21.5 % dried extract by weight of the dried herb. The CRE was composed of 554.86 + 14.65 mg/g berberine (Ber), 60.6 + 0.5 mg/g coptisine (Cop) and 51.9 + 0.2 mg/g palmatine (Pal). Figure 1 shows representative high-5 performance liquid chromatogram of CRE and mixed standard compounds (MSC). The numbers in figure 1 represent the following compounds: [0035] 1 coptisine, [0036] 2 palmatine [0037] 3 berberine.

[0038] The antioxidant activity of Ber, Cop, Jat, Pal, NAC and trolox against free radicals was determined by using the DPPH-assay. Cop, Jat and Pal showed no significant change in DPPH absorption whereas Ber, NAC and trolox showed a strong concentration dependent reduction of DPPH absorption (P<0.01; Fig. 2). Fig. 2: DPPH-scavenging activ-ity of Ber, Cop, Jat, Pal, NAC and Trolox. Results represent the mean DPPH absorption ± SEM normalized to DPBS control of three independent experiments performed in triplicate. * and ** indicates significant differences in comparison to DPBS control; P<0.05 and P<0.01, respectively.

[0039] Figure 3 shows the results of peroxyl radical scavenging activity that was measured with the ORAC-Assay. The results showed that 1 ag of Ber, Cop, Jat and Pal has an antioxidant activity equal to 0.19 ± 0.01 jug, L87 ± 0.07 ag, 1.73 ± 0.08 pg and 0.04 ± 0.002 ag trolox, respectively (Fig. 3). Fig. 3: Trolox equivalent antioxidant capacity (TEAC) of Ber, Cop, Jat and Pal. Results are expressed as pg trolox equivalent/ mg single component ± SEM of four independent experiments performed in triplicates. ** P<0.01 vs. Ber and ## P<0.01 vs. Pal.

[0040] To investigate the neuroprotective effect of CR main alkaloids against t-BOOH induced toxicity, it was examined whether those single components exhibit any cytotoxic effect in the concentration range between 0.1 and 40 MM. Figure 4 shows the effect of CR main alkaloids on cell viability in SH-SY5Y cells. The cells were pre-treated for 24 h with (0-40 MM) of Ber (A), Cop (B), Jat (C) and Pal (D) before the cells were exposed for 2 h either to medium (medium control) or 100 aM t-BOOH (t-BOOR control). Results repre-sent mean cell viability ± SEM of four independent experiments conducted in triplicates. * P<0.05, ** P<0.01 vs. medium -or t-BOOH control.

[0041] It can be taken from figure 4 that there is no significant cytotoxic effect of Ber, Cop, Jat and Pal in comparison to the medium control (P<0.0 I; Fig. 4). Treatment of cells with 100aM t-BOOH resulted in a significant decrease of cell viability to 53.2 ± 1.7 % (P<0.01, vs medium control). Pretreatment of the cells for 24 h with Ber, Jat and Pal (0.140 aNI) before oxidative stress was induced with t-BOOH showed no significant protective effect compared to the t-BOOH control (Fig. 4 A,C and D). However, pretreatment with 1- 40p.IVI Cop for 24 h resulted in a significant increase of cell viability from 53.2 + 1.7 % (tBOOH control) up to 65.6 + 2.6 % (40 p.NI; Fig. 4 B).

[0042] Previously, it was possible to show that 24 h pretreatment with the watery extract of CR had a significant protective effect against t-BOOH induced oxidative stress, with pg/m1 being most effective. To investigate if Cop is responsible for the previously reported protective Effect of CRE, the neuroprotective effect of 24 h pretreatment with CRE (100 pg/ml) was compared with Cop (20 uiM). Figure 5 shows the effect of CRE and Cop on cell viability in SH-SY5Y cells. Cells were pretreated for 24 h with 100 mg/m1 CRE or 20 pM Cop. ** P<0.01 vs. medium -or DMSO control. ## P<0.01 vs. t-BOOHmedium or t-BOOH-DMSO. + P<0.05 vs. t-BOOH-Cop.

[0043] Result revealed that CRE increased cell viability to 71.9 + 2.3 % and Cop to 64.6 + 1.9 % (Fig. 5). Statistical analyses showed that CRE is significantly more effective than Cop (P<0.05). However, it is possible to reach 80 % of the cell viability increase by Cop alone (Fig. 5). Thus, Cop seems to be the main active component for the neuroprotective effect of CRE against t-BOOH induced oxidative cytotoxicity.

[0044] To investigate the antioxidative effect of CRE and Cop on t-BOOH induced oxi-dative stress the amount of reactive oxygen species in the cell were measured with the 2',7'-dichlorodihydrofluorescein diacetate probe (H2DCFH-DA). The mean DCF fluorescence of medium control cells was set to 100 %. Figure 6 shows the effect of CRE and Cop on ROS level in SH-SYSY cells that was determined by H2DCF-DA staining. Results are expressed as DCF fluorescence ± SEM normalized to the medium control of three inde-pendent experiments, which were conducted in triplicate. ** P<0.01 vs. control. + P<0.05 vs Cop.

[0045] Results showed that both CRE and Cop had no effect on the baseline DCF fluorescence. Two hour exposure to 100 pM t-BOOH produced a significant increase in DCF fluorescence to 252.1 + 6.7 % (P<0.01; t-BOOH control). Furthermore, results revealed that only CRE significantly reduced mean DCF fluorescence to 227.7 + 2.5 % (P<0.5 vs tBOOH control; Fig. 6).

[0046] In order to investigate the effect of CRE and Cop on t-BOOH induced apoptosis the morphology of the nucleus was analysed. Figure 7 shows the effect of 100 p.M CRE or 20nM Cop on t-BOOH-induced apoptosis. Results represent the percentage of apoptotic nuclei ± SEM of 3 independent experiments. At least 1600 cells were analysed in each group. ** P<0.01 in comparison to the control (no treatment); ## P<0.01 vs t-BOOH-medium or t-BOOH-DMSO and ++ P<0.01 vs t-BOOH-Cop.

[0047] Results showed that the apoptotic rate in both medium and DMSO control groups was 5.3 + 0.5 % and 6.2 + 0.6 %, respectively. Two hours of t-BOOH treatment increased apoptotic rate in the medium -and DMSO control group significantly to 46.3 + 1.2 % and 47.1 + 1.5 % (P<0.01; Fig. 7). Twenty-four hour pretreatment with CRE (100µg/ml) or Cop (20 tfM) reduced apoptosis significantly to 28.0 + 1.3 % and 36.5 ± 1.4 %, respectively (P<0.01). Statistical analysis revealed that CRE attenuates t-BOOH induced apoptosis significantly more than Cop (P<0.05).

[0048] Since, it was previously reported that oxidative stress induced by t-BOOH leads to opening of the MPT pore and loss of mitochondrial membrane potential (Nieminen et al., 1995; Nieminen et al., 1997; Zhao et al., 2005), we investigated if CRE or Cop could attenuate the loss of MMP in SH-SY5Y cells. Figure 8 shows the effect of CRE and Cop on MIMP. Cells were pretreated for 24 h with CRE (100 µg/m1) or 20 mM Cop before t-BOOH (100 RIVE 2 h) treatment. The mean fluorescence signal of medium control cells was set to 100 %. Results represent mean MMP ± SEM of 3 independent experiments at least 1600 cells were analysed in each group. ** P<0.01 vs. control; ## P<0.01 vs t-BOOH-medium or t-BOOH-DMSO and ++ P<0.01 vs t-BOOR-Cop.

[0049] Results showed that 2h exposure of SH-SY5Y cells with 100 nM t-BOOR significantly reduced the fluorescence signal to 44.2 ± 2.1 % (t-BOOH-medium-control) and 49.2 + 1.5 % (t-BOOH-DMSO-control) in comparison to the medium control (P>0.01), which was set to 100 % (Fig. 8). Pretreatment of the cells for 24 hours with 100 µg/ml CRE or 20 pA4 Cop significantly increased compared to the t-BOOH-medium-control the MMP to 78.2 + 4.3 % and 67.9 + 1.7 %, respectively (P<0.01). Comparison between the CRE and Cop group showed that CRE was more effective than Cop (P>0.05).

[0050] QRT-PCR results confirmed that treatment of the cells with CRE or Cop leads to a significant down-regulation of TXNIP compared with the control-group to 49.5 + 3.8 °A and 71.9 + 3.9 %, respectively (P<0.01; Fig. 9). A comparison between CRE-and Cop-group revealed that the down-regulation of TXNIP was significantly pronounced in the CRE-group (P<0.05).

[0051] Figure 9 shows the relative gene expression ratio (rER) of TXNIP. Results represent mean rER ± SEM of three independent experiments preformed in triplicates. Data were normalized to the non-treatment control which was set to 100 °A. * indicates P<0.01 vs control; + P<0.05 vs. Cop.

[0052] Figure 10 shows TXNIP protein concentration of 1 mg extracted total protein. Results represent mean TXNIP concentration + SEM of three independent experiments preformed in duplicates. * P<0.05, ** P<0.01 in comparison to the control; and ## P<0.01 vs Cop.

[0053] Results of the TXNIP protein expression revealed furthermore that the TXNIP concentration in the control-, CRE-and Cop-group was 1189.2 + 86.6 pg/mg total protein 689.4 ± 53.2 pg/mg total protein and 958.7 ± 70.4 pg/mg total protein, respectively (Fig. 10). Statistical analysis showed that both CRE and Cop reduced the TXNIP protein con-centration significantly compared to the control (P<0.01 and P<0.05) and that CRE was more effective than Cop (P<0.01).

[0054] Figure H shows the results of investigating the neuroprotective effect of CRE and its main alkaloids on MPP+ induced neurotoxicity in SH-SYSY cells. It was analysed if CRE or its main alkaloids exhibit any cytotoxic effect in a concentration range between 6.25 and 50 p1V1 for CRE and 0.1 and 20 pM for Ber, Cop, Jat and Pal, respectively. Results showed that neither CRE nor one of the tested single components showed any significant cytotoxic effect in comparison to the medium control. Twenty-four hour treatment with MPP+ reduced cell viability significantly to 57.1 + 1.4 (14) compared to the medium control which was set to 100 % (P<0.01; Fig. 11).

[0055] Pretreatment of the cells for 24 h with CRE, Ber, Cop, Jat or Ber/Cop significanly increased cell viability to 77.7 + 2.4 (25 pg/m1), 67.2 + 0.9 (10 pM), 66.3 + L7 (5 pM), 65.2 + 1.3 (20 MM) and 71.1+2.7 (5 ttNI Cop and 10 ttNI Ber) in comparison to the MPP+ control, respectively (P<0.01, Fig. 2). However, statistical analysis revealed that pretreatment with CRE was more effective than using Cop, Ber and Tat (P<0.01) and that there was no significant difference between CRE and Ber/Cop. Twenty-four hour pretreatment with Pal did not increase cell viability compared to MPP+ control (Fig. 11). Results repre-sent mean cell viability + SEM of four independent experiments conducted in triplicates. *P<0.05, **P<0.01 vs. medium -or MPP+ control.

[0056] The influence of CRE on MPP+ induced apoptosis was investigated by analysing the morphology of Hoechst 33342 stained nuclei. Results represent the percentage of apop-totic nuclei + SEM of 3 independent experiments. At least 1600 cells were analysed in each group. ** P<0.01 in comparison to the control (no treatment); and ## P<0.01 vs MPP--medium. Results revealed that 24h treatment with MPP+ significantly increased apoptotic rate from 4.2 + 0.3 % in the medium control group to 13.8 + 1 %. Twenty-four hour pretreatment significantly attenuated apoptosis to 8.2 + 0.6 % in comparison to MPP+ control group (P<0.01; Fig. 12).

[0057] The rotarod test was used to test the motor coordination. Results represent the mean percentage + SEM of the time the mice spend on the rod. Data normalized to the PBS control. ** indicates significant difference at P<0.01 vs. PBS control and # indicates P<0.05 vs MPTP control (Fig. 13). Treatment of mice with MPP+ reduced the performance on the rotating spindle to 59.3 + 9.6 % in comparison to the PBS control group (P<0.01; Fig. 13). Treatment of the animals with 5, 10 and 20 mg/kg CRE resulted in a dose dependent improvement of the motor coordination to 66.2 ± 8.3 %, 80.7 ± I L9 % and 94.3 + 12.5 %, respectively. Statistical analyses revealed that treatment with 20 mg/kg CRE significantly reduced MPP+ induced interruption of the motor coordination to the level of PBS control (P<0.05 vs MPTP control). However, treatment of the mice with 20 mg/kg CRE without MPTP showed that CRE has no significant influence on the rotarod test performance.

[0058] To further test the effect of CRE on mouse movement disorder induced by MPTP we conducted the pole-test. Figure 14 shows the effect of CRE on movement disorders measured by the pole test. Results represent the mean time the mice needed to turn completely (t-turn, A) and the total time the animal required to climb down the pole (t-sum, B).

Black bars represent mean time + SEM before MPTP or PBS control treatment and the gray bar shows the results after treatment. * and ** indicates significant differences vs PBS control [P<0,05 and 0.01, respectively). ## indicates P<0.01 vs MPTP control. Treatment of mice with MPTP significantly increased t-turn and t-sum from 1.5 + 0.1 s and 7.4 ± 0.3 s (PBS control) to 2.2 + 0.1 s and 9 + 0.5 s, respectively (P<0.01; P<0.05; Fig. 14). Treat- ment with 5, 10, 20 mg/kg CRE showed a dose dependent reduction of t-turn to 2.1 + 0.1 s, 1.9 ± 0.2 s and 1.7 + 0.1 s (P<0.01) as well as t-sum to 8 + 0.3 s, 7.2 + 0.3 s (P<0.01) and 7.1 + 0.4 s (P<0.01) in comparison to MPTP control group, respectively. Further analysis revealed that 20 mg/kg CRE abolished completely MPTP induced movement disorder in-duced by MPTP (P<0.01). Results from CRE control group revealed that treatment with CRE showed no significant influence on t-turn but t-sum was even better than in the PBScontrol-group (P<0.05).

[0059] To measure the degeneration of dopaminergic neurons in the SN pars compacta and ventral tegmental area (VTA) we used stereology method to count the number of TH+ cells in each area. Results shown in figure 15 represent the mean number of TH+ cells + SEM in the SN (A) and VTA (B). ** indicates significant difference of P<0.01 vs. PBS control and # indicates P<0.05 vs MPTP-control. Results revealed that MPTP treatment significantly reduced the number of TH+ cells from 12183.53 ± 1146.74 TH+ cells (vehi-cle control) to 6900 + 815.07 cells in the SNpc (P<0.01). The results showed furthermore that additional treatment of the mice with CRE increased significantly the number of TH+ neurons in SNpc to 9980.30 ± 542.29 TH+ cells/SN in comparison to the MPTP control (P<0.05; Fig. 15). Statistical analysis revealed furthermore that there was no significant difference between vehicle control and the CRE treatment group. However, there was no difference between the numbers of TH+ cells in the striatum detectable between the exam-ined groups (Fig. 15).

Detailed Description of the invention

[0060] The present disclosure provides a compound or a mixture of compounds for the treatment of neurodegenerative diseases or injuries caused by oxidative stress.

[0061] Neurodegenerative diseases are defined as hereditary and sporadic conditions that are characterized by progressive nervous system dysfunction. These disorders are often associated with atrophy of the affected central or peripheral structures of the nervous system. They include diseases such as Alzheimer's disease, dementias, Brain Cancer, Degenerative Nerve Diseases, Encephalitis, Epilepsy, Genetic Brain Disorders, Head and Brain Malformations, Hydrocephalus, Stroke, Parkinson's Disease, Multiple Sclerosis, Amyo- trophic Lateral Sclerosis (ALS or Lou Gehrig's Disease), Huntington's Disease, Prion Dis-eases and others.

[0062] The term "oxidative stress" designates an imbalance between the systemic manifestation of reactive oxygen species (ROS) and a biological system's ability to readily de- toxify the reactive intermediates or to repair the resulting damage. Imbalances in the nor-mal redox state of cells can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA. Further, some ROS act as cellular messengers in redox signalling. Thus, oxidative stress can cause disruptions in normal mechanisms of cellular signalling.

[0063] In humans, oxidative stress is thought to be involved in the development of cancer, Parkinson's disease, Alzheimer's disease, atherosclerosis, heart failure, myocardial infarction, fragile X syndrome, Sickle Cell Disease, lichen planus, vitiligo, autism, infection. chronic fatigue syndrome and diabetes.

[0064] Modulating the activity of a protein or enzyme means to up-or down-regulate its functional properties. Such a modulation can be based on direct or allosteric interaction with the respective protein as well as on suppression or down-regulation of cellular protein concentrations.

[0065] The neuroprotective effect of CRE and its main compounds against t-BOON and MPPEMPTP induced cytotoxicity was investigated in-vitro and in-vivo. CR is a very important herb in Chinese medicine, which is used in many different herbal prescriptions. TBOOH has been extensively used in the last decades to induce oxidative stress in cell cul-ture experiments. It is well known that tert-butoxyl radicals, which are produced by iron dependent conversion of t-BOOH, increase intracellular ROS production and evoke lipid peroxidation (Nieminen et al., 1997; Amoroso et al., 1999; Martin et al., 2001; Annunziato et al., 2003).

[0066] The results confirmed that two hour treatment of SH-SY5Y cells resulted in a significant cell viability reduction with a strong dose-dependency. Although Ber captures free radicals in a concentration-dependent manner and Jat showed a strong peroxyl radical scavenging capacity, there was no attenuation of the cell viability loss detectable by pre-treatment with different concentrations of Ber, Jat and Pal (Fig. 2, 3).

[0067] However, it turned out that pretreatment with Cop resulted in a dose dependent reduction of t-BOOH induced cytotoxicity and decreased significantly cell viability loss. A comparison with the cytoprotective effect of CRE, which was reported previously (Friedemann et al., 2014), revealed that pretreatment with Cop in a comparable concentra-tion as in the CRE achieved approximately 80% of the effect of the CR watery extracts.

[0068] This suggests that Cop alone is the main component, but not the only compound responsible for the neuroprotective effect. So far the extract of Coptis chinensis Franch.

has been shown to include the following compounds: magnoflorine, groenlandicine, ber-berastine, demethyleneberberine, lycoranine B, jatrorrhizine/columbamine, epiberberine, coptisine, thalifendine/berberrubine, palmatine, berberine and dihydrochelerythrine (Chuang et al.,1996; Ding et al., 2012; Jiang et al., 2014; Wang et al., 2004;).

[0069] As shown in Fig. 1, we used just 4 alkaloids of the extract namely berberine, cop-tisine, jatrorrhizine and palmatine. Therefore, we hypothesize that the cytoprotective effect of CR against t-BOOH induced oxidative stress could be imitated by the combination of two or more single compounds.

[0070] It was reported previously by Drahota et al. (2005) that treatment with t-BOOH leads to a reduction of the v1MP and increases apoptosis in-vitro. Therefore, it was tested if Cop pretreatment attenuates the reduction of the MMP as well as the increased apoptotic rate and compared the results with the effect of the CRE. Results revealed that pretreatment of SH-SY5Y cells with Cop or CRE improved the MMP compared to the t-BOOH control and that the CRE was more effective than Cop. The same applies also to the reduc-tion of t-BOOH induced apoptosis by Cop and CRE. These findings strengthen furthermore the hypothesis that the cytoprotective effect of the CRE could only be explained by a combination of different single compounds. However, Cop seems to be the main active component, which protects the cells against t-BOOH induced oxidative stress.

[0071] However, these results suggest that the cytoprotective effect is at least partly achieved by improved mitochondrial function and reduction of apoptosis.

[0072] Based on the strong peroxyl radical scavenging activity of Cop one might come to the hypothesis that Cop protects the cells from t-BOOH induced oxidative damage by reducing oxidative stress directly. However, this hypothesis contradicts the fact that Tat also showed a high affinity to capture peroxyl radicals without to have an impact on cell viability. In a previous study we could show that the cytoprotective effect of CRE was independ-ent of its antioxidative properties (Friedemann et al., 2014). Those findings lead us to the hypothesis that the cytoprotective effect of Cop was independent of its antioxidative properties.

[0073] To prove this hypothesis the antioxidative effect of CRE and Cop with the cell-based H2DCFH-DA assays was investigated. The results showed that only the CRE showed a slight antioxidative effect. However, this effect was too small to explain the present neuroprotective effect of the CRE. The results are in good agreement with our hypothesis and suggest that the amount of anti-oxidative compounds in the extract or the concentration of Cop was too low to have a significant influence on the ROS generation induced by the rather high dose of t-BOOH.

[0074] Previously it was reported that genome-wide transcriptome analysis on Human Genome U219 microarrays (Affymetrix, SantaClara, USA) of SH-SY5Y cells revealed that 24 hour treatment with CRE significantly regulated only the expression of two genes; MTND1 and TXNIP (Friedemann et al., 2014). In this work it was hypothesized that down-regulation of TXNIP, a 50-kDa protein which belongs to the a-arrestin protein family, could be at least partly responsible for the neuroprotective effect of CRE on t-BOOH induced oxidative damage. It is well known that TXNIP is an inhibitor of thioredoxin (Trx), which is essential for intracellular responses to ROS (Nordberg et al., 2001; Ma, 2010; Lu and Holmgren, 2012). In case of oxidative stress TXNIP binds to Trx and pre-vents that Trx plays its major role as anti-oxitant, in DNA synthesis and repair, in cellular signaling and transcription control and in inhibition of apoptotic pathways (Lu et al., 2007, 2012; Saitoh et al., 1998; Saxena et al., 2010; Nordberg et al., 2001; Ma, 2010; Lu and Holmgren, 2012).

[0075] Therefore, down regulation of TXNIP by CRE or Cop could reduce the amount of inactivated Trx and this results in an attenuation of apoptotic signaling in the cell (AlGayyar et al., 2011; Gao et al., 2013). This suggests that down regulation of TXNIP could protect SH-SYSY cells from apoptosis, improves DNA repair as well as cellular signaling, strengthen oxidative defense system during high oxidative stress. Our results revealed that both CRE and Cop decreased TXNIP mRNA and protein concentration. However, the reduction of TXNIP expression should have an impact on cellular ROS-level. Yoshihara et al. (2010) reported that down-regulation of TXNIP attenuates the inhibition of the Trx re-dox cycle by TXNIP and this results in a reduction of cellular oxidative stress. Since our results showed only a small reduction of ROS by CRE and no effect of Cop, one might speculate that the present down-regulation of TXNIP was not sufficient enough to have a significant influence on the ROS-level generated by the relatively high t-BOOH concentration used in this study. Gao et al. (2013) showed that inhibition of TXNIP expression by activation of AMP-activated protein kinase protects podocytes from oxidative stress in-duced injury, without any influence of the cellular ROS-level. They hypothesized that a reduction of TXNIP expression leads to an attenuation of the ASK1-P58 signaling pathway which protects the cells from oxidative stress induced injuries. Nevertheless, further studies should clarify if down-regulation of TXNIP by CRE and Cop also leads to a attenuation of the ASK1-P58 signaling pathway in SH-SYSY cells and whether this mechanism is involved in the neuroprotective effect of CRE and Cop against oxidative stress induced cytotoxicity.

[0076] In conclusion we showed that 2 h treatment of SH-SY5Y neuroblastoma cells with 100pM t-BOOH caused an increased intracellular ROS, reduced the MNIP, increased apoptosis and as a consequence lowered cell viability. Treatment with CRE and one of its main alkaloids Cop attenuated all those effects of t-BOOH besides intracellular ROS level.

[0077] Further analysis showed that CRE was more effective than Cop. However, we 30 could achieve approximately 80% of the effectiveness of CRE by treatment with Cop. This leads us to the amazing conclusion that Cop is the main active single component of CRE for the prevention of t-BOOH induced oxidative stress cell injury.

[0078] This research work provided evidence that CRE and Cop, one of its main single components, might have potential therapeutic value for the treatment of neurodegenerative disease associated with oxidative stress, like Alzheimer's disease or Parkinson's disease.

[0079] The PD is one of the most common neurodegenerative diseases (Tanner and Goldman, 1996; Miller et al., 2009). Although many efforts have been made in the recent years the underlying mechanisms of PD are not completely revealed and treatment strategies still tent to suppress the symptoms rather than trying to attenuate or stop the progression (Ossig and Reichmann, 2013; Pedrosa and Timmermann, 2013). Due to the lack of treatment options with good prospects for a long-term improvement the search for new treatment methods and novel therapeutics appears to be essential (Harikrishna et al., 2014).

[0080] By now, there are many different cellular and animal models available to study the effect of pharmaceuticals on the progression of the PD (Cheng et al., 2009; Koch et al, 2009; Takeuchi et al., 2009; Hsieh and Chiang, 2014; Patil et al., 2014). Since PD is char-acterized by the destruction of dopaminergic neurons in the substantia nigra, the use of dopaminergic cell lines became popular to initially study the effect of novel therapeutics on the degeneration of dopaminergic cells. In this study we used the SH-SY5Y neuroblastoma cell line as a model for dopaminergic neurons [Xie et al., 2010]. It is well known that SH-SY5Y cells are able to synthesize dopamine as well as noradrenalin and that they ex-press dopamine transporters (DAT) [Takahashi et al, 1994]. DAT is specifically expressed in dopaminergic neurons in the central nervous system and required for the uptake from MPP+ into neurons [Gainetdinov et al., 1997; Kitao et al., 2007]. Therefore, SH-SY5Y cells are widely used for the MPP+ induced PD model. Although SH-SY5Y cells could be differentiated for example by the use of retinoic acid into a more distinct model of dopa-minergic neurons, we used undifferentiated SH-SY5Y cells, because it was reported that differentiation in this cell type increases tolerance against neurotoxic agents and is not useful anymore to investigate the influence of pharmaceuticals on neuroprotection [Cheung et al., 2009]. Our results revealed that 24 h treatment of SH-SY5Y cells with MPP+ decreases cell viability, induces apoptosis and that those effects could be attenuated by pretreatment of the cells with CRE. Furthermore, we could show that Cop and Ber protect SH-SY5Y cells from MPP+ induced toxicity too, but the neuroprotective effect was less pronounced as seen for the CRE. However, a combination of Ber and Cop lead only to a smal gain of the effect. Those results indicate that Cop and Ber are the main active components in the CRE and that they might act via the same mechanism of action, because there was no real additive or synergistic effect visible when cells were treated with Ber/Cop.

[0081] In the last decade, there were many reports on potential therapeutics which showed a good neuroprotection in-vitro but failed in in-vivo studies. Therefore, we used the MPTP mouse model to confirm our in-vitro results. There are several animal models available to test potential neuroprotective drugs in-vivo [Patil et al., 2014]. Application of MPTP for several days counts as valid method to induce a chronic PD model in C57BL/6 mice [Jackson-Lewis and Przedborski, 2007; Meredith and Rademacher, 2012]. Intraperi-toneally applied MPTP crosses the blood brain barrier and gets oxidized to MPP+ by monoamine oxidase B [Przedborski et al., 2000]. MPP+ is selectively taken up by dopaminergic cells via the dopamine transporter and induces neurodegeneration especially in the substantia nigra [Mayer et al., 1986; Chan et al., 1991]. It was reported previously that MPP+ uptake leads to increased oxidative stress [Hasegawa et al., 1990; Zang and Nrisra, 1993], inhibition of the mitochondrial respiration complex 1 activity, reduced ATP availability and leads to a disruption in Ca++ homeostasis [Nicklas et al., 1985; Mizuno et al., 1987; Kupsch et al. 1995; Davey and Clark, 1996; Chan et al., 2007]. Selective degeneration of dopaminergic neurons in the substantia nigra by NIPTP results in an impaired motor control [Meredith and Kang, 2006; Lee et al., 2013]. Therefore, we investigated the effect of CRE on MPTP induced movement disorders by the rotarod-and pole-test. Results revealed that treatment with CRE improved the motor function in both test in a concentration dependent manner. Those findings leading to the presumption that CRE protects dopaminergic cells from MPTP induced toxicity and thereby improves the dopamine-level in the central nervous system. To test this hypothesis we investigated the effect of CRE on dopamin-ergic cells in the SN and VTA. Stereological analysis showed that treatment with CRE indeed increases the amount of TH+ cells in comparison to the MPTP control group. Since the synthesis of dopamine and other catecholamine neurotransmitters is limited by the activity of TH, loss of TH positive cells reflects a reduction of dopaminergic neurons as it is seen in patients with PD [Haavik and Toska, 1998]. Our results showed that CRE attenu- ates the MPTP induced loss of TH+ cells and inproves motor function in mice. These find-ings are in good agreement with a study conducted by Lo et al. (2011). They reported that the Chinese herbal formula San-Huang-Xie-Xin-Tang (SHXT), which is composed of Coptidis rhizoma, Scutellariae radix, and Rhei rhizome protects C57BL/6 mice from MPTP induced neurotoxicity via its antioxidative and antiapoptotic properties. Coptidis chinensis Franch. is also included in many different Chinese herbal formulas which showed already its beneficial effect in several clinical PD studies suggesting that CR is essential for the treatment of patients with PD [Wang et al., 2012].

[0082] In conclusion we showed that 24h pretreatment with CRE protects SH-SYSY neu-roblastoma cells and C57BL/6 mice from MIPP+/NIPTP induced toxicity in a dose dependent manner, respectively. Cell culture experiments revealed that Ber and Cop might be the main active single components of the CRE responsible for the present neuroprotective effect.

Experimental procedures [0083] The invention will be described by experimental procedures without being limited to the disclosed embodiments.

[0084] Drugs and reagents like 2,2-Di(4-tert-octylphenyl)-]-picrylhydrazyl (DPPH), 2,2 Azobi s (2-methyl propi onami di n e)di hydrochl ori d (AA PH), Trolox, Tert-butyl -hydroperoxide (t-BOOH) and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma (Taufkirchen, Germany). 2',7'-dichlorodihydrofluorescein diacetate (H2DCFH-DA), Mitotracker Red CMX Ros and Hoechst 33342 were obtained from Life Technologies (Darmstadt, Germany). Berberine (Ber), coptisine (Cop), jatrorrhizine (Jat) and palmatine (Pal) were ordered from Cfm Oskar Tropitzsch (NIarkdredwitz, Germany). All other reagents were purchased from Roth (Karlsruhe, Germany).

[0085] Coptis chinensis Franch. was obtained from China Medica (Ch. B. 930034; 83684 Tegernsee, Germany) as dried rhizome. Identity and purity was confirmed according to the Pharmacopoeia of the People's Republic of China (Chinese Pharmacopeia Commission, 2005). Sebastian Kneipp research laboratory for residue analysis and organic trace analysis (Bad Worishofen, Germany) confirmed that heavy metal, pesticide, and microbiological contamination was below the guideline of the Pharmacopoeia Europaea (European Phar-macopoeia Commission, 2014) and Regulation (EC) No 396/2005 of the European Commission.

[0086] Coptis chinensis Franch. extract was prepared by boiling 10 g of grounded rhizome in 100m1 distilled de-ionized water (DDW) for 30 minutes and the extract was centrifuged afterwards (Friedemann et al., 2014). Supernatant was collected and the residue was extracted a second time with 100 ml DDW. Combined supernatants were dried with a rota-ry-vacuum evaporator (60°C, 200 mbar; Rotavapor-R, Btichi) and a vacuum concentrator (Bachofer). Dried extracts were stored at -20°C until use.

[0087] HPLC-Analysis was performed according to the method described previously by Friedemann et al. (2014). Briefly, chromatographic separation was conducted on an Allti-ma C18 (250 mm X 4.6 mm x 5 i_tm, S/N: 213100139, temperature: 25°C) column with 0.1 % trifluoroacetic acid (A) and acetonitrile (B) as mobile phase and a flow rate of 1 ml/min. Berberine (Ber), coptisine (Cop), jatrorrhizine (Jat) and palmatine (Pal) were used as reference standard compounds.

[0088] The DPPH assay was used to measure antioxidative activities of Ber, Cop, Jat and Pal against free radicals (Turkmen et al., 2006; Sharma et al., 2009). Briefly, 5 mM stock solutions of Ber, Jat and Pal were prepared with DDW and for Cop (5 mM) with DMSO. Stock solutions were further diluted with DDW to a final concentration of 0.5 pM -500 tiM and 50 ttl were added to each well of a 96-well-plate. Afterwards 200 pl DPPH (75 p.M) was added to each well and agitated for 5 minutes. Absorbance was measured 3 times at 531 nm (Thermo Multiskan SPECTRUM microplate spectrophotometer) 30 minutes after DPPH was added. Experiments were repeated three times in triplicates.

[0089] The antioxidant capacity of Ber, Cop, Jat and Pal was determined by using the ORAC assay. The ORAC assay was carried out according to the method described by Huang et al. (2002) and Gillespie et al. (2007). Briefly, 225 pl of 10 nM fluorescein solution dissolved in a 75 mM sodium phosphate buffer (SPB; pH 7.4) was pipetted into the well of a 96-well micro-plate and 37 p.1 of sodium phosphate buffer (blank), Trolox (20-80 tiM) or Ber, Cop, Jat, Pal (0.1-80 pM; sample) were added in different concentrations. For kinetic readings 485 nm was used as excitation wavelength and 582 nm as emission wavelength. Fluorescence signals were recorded every 5 minutes for a total time period of 120 minutes.

[0090] Human neuroblastoma SH-SY5Y cell were cultivated in RPMI 1640 medium containing 10 % fetal calf serum, 100 U/ml penicillin and 100 pg/m1 streptomycin. Cells were grown in a humid atmosphere of 5 917 CO2 and 95 % air at 37°C. All cell culture reagents were obtained from Sigma (Taufkirchen, Germany) [0091] Cell viability was determined by the MTT assay as described previously (Friedemann et al., 2014). Ber, Cop, Jat and Pal stock solutions were diluted in medium to their final concentration, steril filtrated and different concentrations were added at the start of the incubation time for 24 h. Afterwards cells were incubated for two hours with 100 p.M t-BOOH. Medium containing t-BOOH was removed, cells were washed with DPBS and 1 mM MTT solution was added for 2 h. Subsequently, 100 pl 2-Propanol was added and the plate was agitated for one hour at 450 rpm at RT. Absorption was measured 3 times with at 570 nm.

[0092] Alternatively 2*104 SH-SY5Y neuroblastoma cells were seeded into the well of a 96-well microtiter plate and incubated for 24 hours in a humid atmosphere of 5 % CO2 and 95 % air at 37°C. MPP4 was added to the culture medium and cells were incubated for further 24 h. Cell viability was measured as described by Friedemann et al. (2014) and the optimal MPP concentration for the further experiments was determined. To test the effect of CRE and its main alkaloids on cell viability cells were treated for 24h with CRE, Ber, Cop, Jat or a combination of Ber and Cop in different concentrations. Thereafter, medium was replaced with fresh medium containing 1.5 mM MPP-and cells were incubated for another 24 h before cell viability was measured.

[0093] Reactive oxygen species in the cell were measured with the 24,7"-dichlorodihydrofluorescein diacetate probe (H7DCFH-DA). SH-SY5Y cells were seeded into a 96-well micro-plate (4*104 cells/well) and incubated with 100pg/m1 CRE or 20pM Cop for 24 hours. Afterwards, fresh medium containing 20 pM H2DCFH-DA was added for 30 min at 37°C in the dark. Subsequently, cells were washed with DPBS and 100 pk1 t-BOOH solution was added. Fluorescence was measured every 5 minutes for 120 minutes (excitation 485 nm; emission 528 nm).

[0094] Apoptotic nuclei and mitochondria] membrane potential were measured as described previously (Friedemann et al, 2014). Briefly, cells were stained first with 25 nM Mitotracker Red CMX Ros for one hour followed by 20 minutes fixation with 4 % paraformaldehyde (PFA) and 10 minutes Hoechst 33342 (4 pM) staining. Images were captured with a Leica microscope and an Axiovision camera. To determine MMP the intensity sum of the Mitotracker Red CMX Ros fluorescence was measured for each cell. Apoptosis was detected by analysing the morphology of the Hoechst 33342 stained nuclei. Experi-ments were repeated 3 times and at least 1600 cells were analysed for each group.

[0095] Total RNA was isolated with the RNeasy MINI Kit (Quiagen; Hilden; Germany) according to the instructions of manufacturer's. Reverse transcription was carried out with the high capacity RNA-to-cDNA Kit (Applied Biosystems). For semi-quantitatively analysis LightCycler 480 SYBR Green 1 Master (Roche) and the following human specific primers were used: TXNIP 5' -GATCACCGATTGGAGAGCCC-3' and 5'-TGCAGGGATCCACCTCAGTA-3' , GAPDH 5'-GC ATC TTCTTTTGCGTCGCC-3' and 5'-CCCAATACGACCAAATCCGTTG-3'. Obtained data were analyzed as described previously (Schefe et al., 2006) [0096] Total proteins were isolated with mammalian cell lysis reagent (CellLytic M) according to instructions of the manufacturer and stored at -80°C until use.

[0097] Protein concentration was determined with Roti-Quant following the protocol provided by the manufacturer. Human Txnip protein concentration was measured with the CircuLex Human TXNIP ELISA Kit (MBL international cooperation, Biozol, Germany) following the instruction of the provided protocol. Experiments were repeated three times and each sample was measured in duplicates.

[0098] Sixty eight-week-old male C57BL/6 mice, with a body weight of 25-30g, were purchased from the animal facility of the "Shanghai Research Center For Model Organisms" (SRCMO) four weeks before the experiments and kept and bred at the Research department of the SRCMO. The mice were group-housed with five mice per cage and had free access to food and water. The animal housing facility of the SRCMO had a ambient temperature of 23 ± 1°C, a relative humidity of 60 ± 5% and a 12 hours light/dark cycle. Health condition and bodyweight of each animal was checked every day during the experiment. Cages were changed once per week and 3 days before MPTP treatment [Jackson-Lewis, 2007]. All experiments were strictly conducted in accordance to the institutional and national guidelines for animal experiments.

[0099] Animals were assigned randomly to six different groups. Group 1 mice received phosphate buffer saline (PBS) orally for 7 days, the following 7 days PBS was injected intra-perioneal (i.p.) and orally. Group 2 mice were orally administered with saline for 7 days, from day 8 until day 14 they receive one i.p. injection of 20mg/kg/day MPTP (dissolved in PBS) every 24 hours as well as one PBS injection/day orally [Jackson-Lewis and Przedborski, 2007]. Group 3, 4 and 5 were orally treated with 5, 10 and 20mg/kg/day CRE (dissolved in PBS) for seven days, respectively and from day 8 MPTP was administered as described for group 2 until day 14 together with the oral CRE treatment. The procedure of group 6 was the same as for group 5, with the exception that on day 8 until day 14 PBS was injected instead of MPTP. The maximum injected volume of MPTP and CRE was 0,3 ml. During the experiment the safety guidelines described by Lau et al. (2005) for handling MPTP were strictly followed.

[00100] The rotarod test was used to study the motor status of the mice before and after the treatment. During the rotarod test the mice had to stay upon the turning rod and the motor function is measured by the duration each animal could stay upon the rod. The ex-periments were conducted on a Big Behr-RRTM with a rotating spindle diameter of 2.3 cm. The system allows operating five mice at the same time, whereat the mice were kept in single compartments. The total time a mice stayed on the rotating cylinder was recorded manually by the operator. All experiments were conducted by two operators to ensure a proper work flow and crosschecking of the experimental parameters.

[00101] Four days before the first orally injection of PBS or CRE the animals were trained two times for 10 minutes to walk on the rod, which was turning with a speed of 20 rpm. The time between each training session was 30 minutes. The following day the baseline rotarod test was performed. The retention time on the rod for each mice was recorded three times with 30 minutes rest interval between each run. The mean retention time on the rod of each mice was calculated. Based on those results the cages (5 mice/cage) were per-assigned to the 6 different groups in a way that the difference between the 6 groups in the rotarod test was as small as possible.

[00102] The day after the last orally injection of PBS or CRE, the rotarod test was repeated and the average retention time on the rod was calculated as described above, normalized to the baseline test values for each group and the value of the control group (group I) was set to 100 %.

[00103] Two days before PBS or CRE was injected orally for the first time the mice were trained for the pole test. In the first part of the training session, the mice were placed head-down on top of a rough-surfaced pole (diameter 8 mm; height 55 cm) and had to climb down into their home cage. This exercise was repeated 5 times, afterwards the mice was placed head-up on top of the pole and had first to turn around until there head was facing completely downward, subsequently they had to climb into their home cage again. Also this exercise was repeated 5 times. After 30 minutes of rest the second part of the previous training was repeated again five times. The day after the first pole test was conducted. Three trails were carried out for each animal and the time it took the mice to orientate their head from the head-up position completely downward was recorded (t-turn), as well as the total time it took the mice to descend into the home cage (t-total). If a mice could not turn downward or did not reaches the home cage within 120 seconds the test was stopped and 120 seconds were recorded. Also 120 seconds were taken if the mice fell down from the pole. After the experiment the best time for t-turn and t-total were used to calculate the mean time for each group, which was predefined with the rotarod test and the statistical differences between the six groups were calculated with the t-test. After this calculation the final grouping was arranged in a way that there was no significant difference between each group in the rotarod-test and pole-test for the baseline recording. On the day after the last orally injection of PBS or CRE the pole-test was conducted again as described above.

[00104] TH immunohistochemistry one of the most important methods to detect injury or death of dopaminergic neurons in the substantia nigra and ventral tegmental area in animal models of PD has been used in this study [Haavik and Toska, 1998; Oiwa et al., 2002; Kramer et al., 2007]. All animals were anesthetized with chloral hydrate and the brains were perfused with PBS followed by 4 °.o paraformaldehyde. The brains were then dissected and postfixed in 4 % paraformaldehyde overnight at 4°C. Thereafter, the tissues were dehydrated and embedded in paraffin wax for transportation from Shanghai to Hamburg. Following the transportation the brains were deparaffinized, hydrated and cryoprotected by incubating them in 30 (I'O sucrose overnight. Subsequently, brains were embedded in egg yolk supplemented with 10 % sucrose and 5 % glutaraldehyde. Embetted brains were stored at -80°C until use. For immunohistochemistry, 25 jiM coronal sections were cut with a cryostat, collected free-floating in cryoprotection solution and stored at -20°C until further use. Anti-thyrosine hydroxylase staining was conducted as described by Kramer et al. (2007). Briefly, free-floating sections were stained with primary monoclonal mouse anti-thyrosine hydroxylase antibody (1:2000; DiaSorin, Stillwater, Massachusetts, United States). Diaminobenzidine detection was conducted with the Vectastain ABC Kit (Vector Laboratories, Burlingame, California, United States) according to the instruction of the manufacturer.

[00105] For stereology, TH-positive (TH+) cells in every 6th section were counted in the substantia nigra and ventral tegmental area by stereology. Counting was conducted by using optical fractionator method (Stereoinvestigator, MBF Bioscience) according to the method described by Kowsky et al. (2007) on an Axioskop 2 microscope (Zeiss) with an x63 oil immersion objective. Four animals were analysed for each group.

[00106] Data are presented as means ± SEM of n experiments. Statistical significance between groups was determined with OriginPro 8.5 by ANOVA followed by the BonferroniPost-Hoc-Test. Statistical analysis of the data from pole-test and rotarod-test were deter-mined by two way t-test. P<0.05 was considered as statistically significant.

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Claims (10)

1. Claims
2. 2.
3. 3.
4. 4.
5. 5.
6. 6.

   A composition for the treatment and prevention of neurodegenerative diseases and in-juries induced by oxidative stress comprising coptisine or an extract from Coptis rhizome including pharmaceutically applicable salts, tautomers and stereoisomers of the compound, including mixtures thereof in all ratios.

   The composition of claim 1, wherein coptisine is combined with at least one further compound selected from the group comprising berberine, jatrorrhizine and palmatine including pharmaceutically applicable salts, tautomers and stereoisomers of the compounds, including mixtures thereof in all ratios.

   The composition of claim 1 or 2, wherein the neurodegenerative diseases is selected from the group comprising Alzheimer's Disease and other dementias, Brain Cancer, Degenerative Nerve Diseases, Encephalitis, Epilepsy, Genetic Brain Disorders, Head and Brain Malformations, Hydrocephalus, Stroke, Parkinson's Disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease), Huntington's Disease, Prion Diseases.

   The composition of one of claims 1 to 3, wherein the oxidative stress induced injury is selected from the group comprising cancer, Parkinson's disease, Alzheimer's disease, atherosclerosis, heart failure, myocardial infarction, fragile X syndrome, Sickle Cell Disease, lichen planus, vitiligo, autism, infection, chronic fatigue syndrome and diabetes.

   A method for the use of coptisine or an extract from Coptis rhizome for the treatment of neurodegenerative diseases or oxidative stress induced injuries.

   The method of claim 5, wherein coptisine is combined with at least one further compound selected from the group comprising berberine, jatrorrhizine and palmatine including pharmaceutically applicable salts, tautomers and stereoisomers of the compounds, including mixtures thereof in all ratios
7. 7. The method of one of claims 5 or 6, wherein the neurodegenerative disease is selected from the group comprising Alzheimer's Disease and other dementias, Brain Cancer, Degenerative Nerve Diseases, Encephalitis, Epilepsy, Genetic Brain Disorders, Head and Brain Malformations, Hydrocephalus, Stroke, Parkinson's Disease, Multiple Scle-rosis, Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease), Huntington's Disease, Prion Diseases.
8. 8. The method of claim 7 wherein the neurodegenerative disease is Parkinson's disease.
9. 9. The method of one of claims 5 to 8, wherein the oxidative stress induced injury is se-lected from the group comprising cancer, Parkinson's disease, Alzheimer's disease, atherosclerosis, heart failure, myocardial infarction, fragile X syndrome, Sickle Cell Disease, lichen planus, vitiligo, autism, infection, chronic fatigue syndrome and diabetes.
10. 10. A method for the use of coptisine or an extract from Coptis rhizome for modulating the activity of thioredoxine interacting-protein.