USE OF EARTHWORM PROTEIN FOR PREPARING PHARMACEUTICAL COMPOSITION FOR PROTECTION OF BRAIN NEURONS

Abstract

The present invention relates to a use of earthworm protein. Particularly, the present invention provides a use of earthworm protein for preparing a pharmaceutical composition which can enhance the expression of an antioxidant gene in brain neurons or inhibit the effects of -amyloid protein (A) and 1-methyl-4-phenylpyridinium (MPP.sup.+) on brain neurons. As such, the pharmaceutical composition comprising the earthworm protein has effects of reducing damage to brain neurons.

Claims

1. A use of earthworm protein for preparing a pharmaceutical composition for protection of brain neurons, wherein the earthworm protein does not comprise lumbrokinase and is capable of reducing damage to brain neurons.

2. The use of claim 1, wherein the pharmaceutical composition for protection of brain neurons is used to enhance an expression of an antioxidant gene in neurons.

3. The use of claim 2, wherein the antioxidant gene is SOD1, SOD2 or GPX.

4. The use of claim 2, wherein the pharmaceutical composition for protection of brain neurons has a concentration of earthworm protein of approximately 0.05-0.7 mg/ml.

5. The use of claim 1, wherein the pharmaceutical composition for protection of brain neurons is used to inhibit damage of -amyloid protein (A) to neurons.

6. The use of claim 5, wherein the pharmaceutical composition for protection of brain neurons is used to prevent Alzheimer's disease.

7. The use of claim 5, wherein the pharmaceutical composition for protection of brain neurons has a concentration of earthworm protein of approximately 0.005-0.05 mg/ml.

8. The use of claim 1, wherein the pharmaceutical composition for protection of brain neurons inhibits damage of 1-methyl-4-phenylpyridinium (MPP.sup.+) to neurons.

9. The use of claim 8, wherein the pharmaceutical composition for protection of brain neurons is used to prevent Parkinson's disease.

10. The use of claim 8, wherein the pharmaceutical composition for protection of brain neurons has a concentration of earthworm protein of approximately 0.04-0.08 mg/ml.

11. The use of claim 1, wherein the earthworm protein comprised in the pharmaceutical composition for protection of brain neurons is obtained by extraction from an earthworm with water.

12. The use of claim 11, wherein the earthworm protein comprised in the pharmaceutical composition for protection of brain neurons is obtained by extraction from the earthworm with warm water of approximately 40-50 C.

13. The use of claim 11, wherein the earthworm is Pheretima aspergillum (E. Perrier), Pheretima vulgaris Clen, Eisenia rosea savigny, Eisenia Foetida, Pheretima pectinifera or Pheretima guillelmi.

14. The use of claim 2, wherein the pharmaceutical composition for protection of brain neurons has a concentration of earthworm protein of approximately 0.05-0.7 mg/ml.

15. The use of claim 6, wherein the pharmaceutical composition for protection of brain neurons has a concentration of earthworm protein of approximately 0.005-0.05 mg/ml.

16. The use of claim 9, wherein the pharmaceutical composition for protection of brain neurons has a concentration of earthworm protein of approximately 0.04-0.08 mg/ml.

17. The use of claim 12, wherein the earthworm is Pheretima aspergillum (E. Perrier), Pheretima vulgaris Clen, Eisenia rosea savigny, Eisenia Foetida, Pheretima pectinifera or Pheretima guillelmi.

Description

BRIEF DESCRIPTIONS OF THE DRAWINGS

[0017] FIG. 1 is a diagram showing a result of an antioxidation experiment for an earthworm protein extract according to an embodiment of the present invention.

[0018] FIG. 2 is a diagram showing a result of the effect of an earthworm protein extract according to an embodiment of the present invention on the expression of SOD1 gene.

[0019] FIG. 3 is a diagram showing a result of the effect of an earthworm protein extract according to an embodiment of the present invention on the expression of SOD2 gene.

[0020] FIG. 4 is a diagram showing a result of the effect of an earthworm protein extract according to an embodiment of the present invention on the expression of GPX gene.

[0021] FIG. 5 is a diagram showing a result of inhibition of an earthworm protein extract according to an embodiment of the present invention on damage of A to neurons.

[0022] FIG. 6 is a diagram showing a result of inhibition of an earthworm extract according to an embodiment of the present invention on damage of MPP.sup.+ to neurons.

DETAILED DESCRIPTIONS

[0023] Earthworm protein is an extract extracted by processing earthworms, and may be obtained by using the following method. Since extraction methods of earthworm protein are already well known to a person of ordinary skill in the art, the following extraction method is merely an example and the present invention is by no means limited thereto. Optimal concentrations are as shown below.

Embodiment 1: Preparation of Earthworm Protein

[0024] Earthworms were put into water and stirred in order to wash silt, impurities or mucus off the earthworms, and washing was continued until water from the washing became clear. Before washing, an emetic treatment may be performed on the earthworms, so as to allow the earthworms to expel undigested food or impurities inside their bodies. Afterwards, the washed and cleaned earthworms were put into an ozone solution for sterilization, taken out for washing again and then immersed in warm water of 40-50 C., wherein a ratio of the earthworms to water was 1:20-1:5, preferably 1:12-1:8. After leaving the earthworms in the warm water at 40-50 C. for 12-16 hours, the earthworms were grinded using a grinder. A pulp solution resulted from the grinding was firstly filtered using a 50-100 mesh screen cloth. After a filtered solution was collected, the solution was centrifuged for 8-28 minutes at 6000-14000 rpm (which were adjustable according to the volume of the solution), followed by obtaining and filtering a supernatant thereof and then performing another round of centrifugation as described above, and a supernatant was obtained, giving an earthworm extract, which is the earthworm protein referred to herein. Upon testing, it is found that the earthworm protein does not include lumbrokinase. The solution of the earthworm extract described previously may further be concentrated using an ultrafiltration membrane, or further lyophilized to form a powder to be used for subsequent experiments or uses.

[0025] The aforementioned earthworms may be animals belonging to the class of Oligochaeta in the phylum Annelida, such as Pheretima aspergillum (E. Perrier), Pheretima vulgaris Clen, Eisenia rosea savigny, Eisenia Foetida, Pheretima pectinifera or Pherertima guillelmi, but is not limited thereto.

Embodiment 2: Antioxidation Effect of Earthworm Protein on Brain Neurons

[0026] Firstly, the earthworm protein prepared in Embodiment 1 was formulated with pure water into a solution having a concentration of 0.05-0.7 mg/ml, preferably 0.5 mg/ml.

[0027] A mouse brain neuroma cell strain (Neuro-2a, ATCC, CCL-131) was prepared and cultured in a cell culture solution [Dulbecco's modified Eagle's medium (DMEM), 1% Penicillin-streptomycin (Gibco), and 10% fetal bovine serum (Gibco)]. A 6-well cell culture plate was prepared, and 2 ml of the cell culture solution was planted into each well, such that each well had 1.510.sup.5 cells, and the cells were cultured for 24 hours at 37 C.

[0028] Subsequently, without affecting the adhered cells, the culture solution in each well was removed, and the wells were divided into three groups (2 wells for each group) for experiments, in which Group A was a blank control group, Group B was added with 500 M of H.sub.2O.sub.2 (Sigma), and Group C was added with 500 M of H.sub.2O.sub.2 and 0.5 mg/ml of the earthworm protein extract prepared in Embodiment 1 above. After the above groups were added into the wells together with culture solutions, the cells were cultured for 1 hour at 37 C. Afterwards, 5 g/ml of DCFH-DA (Sigma/SI-D6883-50MG) was further added into each well for reaction for 15 minutes at 37 C. Subsequently, H.sub.2O.sub.2 was allowed to react for 1 hour at 37 C. The cells in each well were then rinsed twice using 1 ml of 1 PBS (Gibco). After rinsing, 200 l of trypsin was separately added into each well for reaction in the dark for 5 minutes. The cell solutions were then removed and placed into 1.5 ml centrifugation tubes and centrifuged for 10 minutes at 400g. The supernatant was removed, and the tubes were rinsed once with 1 PBS and centrifuged again for 10 minutes at 400g. The supernatant was removed again, and cell precipitants were suspended in 1 ml of 1 PBS. Afterwards, a flow cytometry was used to detect fluorescent signals of DCFH-DA at an excitation wavelength (450-490 nm) and an emission wavelength (510-550 nm) respectively. The detected values were input into Microsoft Excel software and statistical significance between the two values was analyzed using Student's t-test, and a result thereof is shown in FIG. 1. In comparison to H.sub.2O.sub.2, the p-value of the group indicated by *is <0.05.

[0029] It can be concluded from the result shown in FIG. 1 that under the condition in which the earthworm protein extract of the present invention is included, the concentration of reactive oxygen species is significantly reduced by approximately 25%, which confirms that the earthworm protein extract of the present invention is capable of decreasing generation of free radicals like reactive oxygen species, thereby reducing damage of the reactive oxygen species to brain neurons.

Embodiment 3: Effects of Earthworm Protein on Expression of Antioxidant Genes in Brain Neurons

[0030] Firstly, the earthworm protein prepared in Embodiment 1 was formulated with a normal saline solution into a solution having a concentration of 0.05-0.7 mg/ml, preferably 0.5 mg/ml. Human skin fibroblast cells (CCD-966sk, BCRC No. 60153) were also prepared, and 2 ml of the cell culture solution was planted into each well of a 6-well plate, such that each well had 1.510.sup.5 cells, and the cells were then cultured for 24 hours at 37 C. Subsequently, without affecting the adhered cells, the culture solution in each well was removed, and the wells were divided into the following groups for experiments, which respectively includes: a blank control group, a group having only H.sub.2O.sub.2 added thereto (H.sub.2O.sub.2 only), and a group having H.sub.2O.sub.2 and 0.5 mg/ml of the earthworm protein extract prepared in Embodiment 1 added thereto (earthworm protein). The cells were cultured for 24 hours at 37 C. before analysis of the expression of specific genes.

[0031] The foregoing groups of cells were recycled, and RNAs were extracted therefrom using an RNA extraction kit (Geneaid), and the RNAs were reversely transcribed into cDNAs using a reverse transcriptase (Invitrogen). Afterwards, by using a Real-Time PCR system (ABI Step One Plus Real-Time PCR system), qPCR (KAPA CYBR FAST qPCR Kits, KAPA Biosystems) was performed using the primers listed in Table 1 respectively, so as to quantify the expression of the following genes: SOD1, SOD2 and GPX. Relative quantitative analyses on gene expression were conducted using a 2-.sup.Ct method. The results related to the expression of the genes are shown in FIGS. 2 to 4, wherein the p-values of the groups indicated by * are <0.05, and the p-values of the groups indicated by *** are <0.001.

TABLE-US-00001 TABLE 1 Sequence Primer Length Product Length Gene Primer Number (ntds) (ntds) SOD1 SOD1-F SEQ ID NO: 1 21 227 SOD1-R SEQ ID NO: 2 20 SOD2 SOD2-F SEQ ID NO: 3 21 116 SOD2-R SEQ ID NO: 4 23 GPX GPX1-F SEQ ID NO: 5 21 105 GPX1-R SEQ ID NO: 6 19

[0032] It can be concluded from the results shown in FIGS. 2 to 4 that, under the condition in which the earthworm protein extract of the present invention is included and left to take effect for 24 hours, a relative value of SOD1 gene expression is increased from 0.66 to 1.99, a relative value of SOD2 gene expression is increased from 0.28 to 1.61, and a relative value of GPX gene expression is increased from 0.17 to 1.35. Therefore, the earthworm protein extract of the embodiment of the present invention can indeed induce increases in expressions of antioxidant genes such as SOD1, SOD2 and GPX.

Embodiment 4: Inhibitory Effect of Earthworm Protein on Damage of A to Neurons

[0033] The earthworm protein prepared in Embodiment 1 was formulated with a normal saline solution into a solution having a concentration of 0.005-0.05 mg/ml, preferably 0.008 mg/ml. A human neuroblastoma cell strain (SH-SY5Y, ATCC CRL-2266) was also prepared, and 2 ml of the cell culture solution was planted into each well of a 6-well plate, such that each well had 210.sup.4 cells, and the wells were divided into the following groups for experiments, which respectively include: a blank control group, a -amyloid peptide (Lifetein) group (A only), a Methyllycaconitine (MLA) control group added with A (PC+A), and a group added with 0.008 mg/ml of the earthworm protein extract prepared in Embodiment 1 and A (earthworm protein+A). The cells were cultured for 24 hours at 37 C. before analysis of survival rates of brain neurons.

[0034] Firstly, 15 l of an MTT (thiazoyl blue tetrazolium bromide, AMRESCO/0793-5G) solution was added into each well, and the cells were cultured for 4 hours at 37 C. The solution was subsequently removed and 50 l of a DMSO (ECHO/DA1101-000000-72EC) solution was added into each well so as to dissolve formazan formed by reaction. The 6-well plate was placed on a shaker and shaken for 10 minutes, and then an ELISA analyzer (BioTek) was used to measure absorbance values at a wavelength of 570 nm, and cell survival rates (%) were calculated using the following formula: (an absorbance value of a test group/an absorbance value of a control group)100%. Finally, the values were input into Microsoft Excel software and statistical significance between the values was analyzed using Student's t-test, and a result thereof is shown in FIG. 5. In comparison to the A only group, the p-value of the group indicated by * is <0.05, the p-value of the group indicated by ** is <0.01; in comparison to the blank control group, the p-value of the group indicated by ### is <0.001.

[0035] It can be concluded from the result shown in FIG. 5 that, under the condition in which the earthworm protein extract of the embodiment of the present invention is included, the cell survival rate is increased from 51.2% to 58.2% and the amplitude of increase is approximately 7%, which proves that the earthworm protein extract of the embodiment of the present invention is capable of inhibiting the effects of A on brain neurons and reducing the damage thereof; therefore, the earthworm protein extract can also be applied in the prevention of Alzheimer's disease.

Embodiment 5: Inhibitory Effect of Earthworm protein on Damage of MPP to Neurons

[0036] The earthworm protein prepared in Embodiment 1 was formulated with a normal saline solution into a solution having a concentration of 0.04-0.08 mg/ml, preferably 0.06 mg/ml. A human neuroblastoma cell strain (SH-SY5Y, ATCC CRL-2266) was also prepared, and 2 ml of the cell culture solution was planted into each well of a 6-well plate, such that each well had 210.sup.4 cells, and the wells were divided into the following groups for experiments, which respectively include a blank control group, an MPP+ group (MPP.sup.+ only), and a group having 0.06 mg/ml of the earthworm protein extract prepared in Embodiment 1 and MPP added thereto (earthworm extract+MPP.sup.+). The cells were cultured for 24 hours at 37 C. before analysis of survival rates of brain neurons.

[0037] Firstly, 15 l of an MTT (AMRESCO/0793-5G) solution was added into each well, and the cells were cultured for 4 hours at 37 C. The solution was subsequently removed and 50 l of a DMSO (ECHO/DA1101-000000-72EC) solution was added into each well so as to dissolve formazan formed by reaction. The 6-well plate was placed on a shaker and shaken for 10 minutes, and then an ELISA analyzer (BioTek) was used to measure absorbance values at a wavelength of 570 nm, and cell survival rates (%) were calculated using the following formula: (an absorbance value of a test group/an absorbance value of a control group)100%. Finally, the values were input into Microsoft Excel software and statistical significance between the values was analyzed using Student's t-test, and a result thereof is shown in FIG. 6. In comparison to the MPP only group, the p-value of the group indicated by *** is <0.001; in comparison to the blank control group, the p-value of the group indicated by ## is <0.01.

[0038] It can be concluded from the result shown in FIG. 6 that, under the condition in which the earthworm protein extract of the embodiment of the present invention is included, the cell survival rate is increased from 74.1% to 97.21%, a significant increase of approximately 23.11%, which proves that the earthworm protein extract of the embodiment of the present invention is capable of inhibiting the effects of MPP on brain neurons and reducing the damage thereof; therefore, the earthworm protein extract can also be applied in the prevention of Parkinson's disease.

[0039] The pharmaceutical composition for protection of brain neurons of the embodiments of the present invention can further be added with carriers or other adjuvants known in the art. The dosage form thereof may be, but is not limited to, solutions, capsules or tablets.

[0040] It can be proven from the above results that the earthworm protein extracts in the pharmaceutical compositions prepared in the embodiments of the present invention have excellent antioxidation effects for brain neurons, and can enhance the expression of antioxidant genes of the neurons and inhibit the effects of A or 1-methyl-4-phenylpyridinium on brain neurons so as to reduce the damage to the brain neurons, and thus can further be used in pharmaceutical compositions for preventing Alzheimer's disease or Parkinson's disease.