Pharmaceutical composition for preventing or treating neurodegenerative diseases which includes flower extract of <i>Daphne genkwa </i>or fractions thereof as active ingredient
11738063 · 2023-08-29
Assignee
Inventors
- Won Gon Kim (Daejeon, KR)
- Baek Soo HAN (Daejeon, KR)
- Jeong Su BYUN (Daejeon, KR)
- Ha Young CHOI (Daejeon, KR)
Cpc classification
A23L33/105
HUMAN NECESSITIES
A61K31/36
HUMAN NECESSITIES
A61K36/83
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to a pharmaceutical composition and health functional food for preventing or treating neurodegenerative diseases which include a flower extract of Daphne genkwa or fractions thereof as an active ingredient. The pharmaceutical composition and health functional food for preventing or treating neurodegenerative diseases are, not like extracts from stems and/or roots of Daphne genkwa, free from genotoxicity and have superior prophylactic or treatment effects against neurodegenerative diseases.
Claims
1. A method of treating or ameliorating a neurodegenerative disease, comprising administering to a mammal in need thereof a composition comprising a therapeutically effective amount of an aqueous extract or lower-alkyl alcohol extract of flower or flower buds of Daphne genkwa or a fraction thereof, wherein the neurodegenerative disease is Parkinson's disease or frontotemporal dementia, and the therapeutically effective amount is 10 mg/kg to 100 mg/kg per day.
2. The method of claim 1, wherein the extract is a C.sub.1 to C.sub.4 alkyl alcohol extract.
3. The method of claim 1, wherein the extract is an ethanol extract.
4. The method of claim 1, wherein the extract comprises Genkwanin N or Yuanhuacine as an active ingredient.
5. The method of claim 1, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
6. The method of claim 1, wherein the composition is a health functional food composition comprising the extract as a food additive.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
(10) Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
Comparative Example 1
Genotoxicity Test of the Extract of Stalk and/or Root of Daphne genkwa
(11) In order to confirm in vitro genotoxicity of the extract of stalk and/or root of Daphne genkwa according to Korean Patent Registration No. 10-1631589 filed by the present applicant, a bacterial reverse mutation assay and a chromosomal aberration test were performed as described below.
(12) 1-1. Production of the Extract of Stalk and/or Root of Daphne genkwa
(13) 14 kg of stem of Daphne genkwa and 6 kg of root of Daphne genkwa were finely cut and immersed in 120 L of 80% ethanol for 48 hours, and filtered to separate it into a solid component and a first liquid component. The separated solid component were again immersed in 120 L 80% ethanol for 24 hours, and filtered to obtain a second liquid component. The obtained first liquid component and the second liquid component were mixed with each other, and the mixture was concentrated under reduced pressure. The residue was freeze-dried, thereby obtaining 1140.9 g of the extract of stalk and/or root of Daphne genkwa (hereinafter referred to as “DG-EX”).
(14) 1-2 Bacterial Reverse Mutation Assay
(15) In order to confirm genotoxicity of the extract of stalk and/or root of Daphne genkwa, a bacterial reverse mutation assay was conducted on the basis of the guidelines of Ministry of Food and Drug Safety by Biotoxtech, a GLP (Good Laboratory Practice) organization.
(16) In order to confirm the genomic mutation induction of DG-EX obtained in the Test 1-1, a bacterial reverse mutation assay for the presence of metabolic activation system (S9+) and non-existence (S9−) was performed with Salmonella typhimurium TA98, TA100, TA1535, TA1537, histidine requiring strain, and Escherichia coli WP2uvrA (pKM101), a tryptophan requiring strain.
(17) (1) Capacity Setting Test
(18) In order to set the maximum dose of the bacterial reverse mutation assay of present test, the recommended dose of 5,000 μg/plate was used as the maximum capacity, and the capacity setting test was carried out at 1,250, 313, 78.1, and 19.5 μg/plate in common rate 4.
(19) As a result, it was found that inhibition of growth by DG-EX was observed on 5,000 μg/plate of TA98 strain, and above 313 μg/plate of TA100, TA1535 and TA1537 strains in the absence of the metabolism activation system, and on 5,000 μg/plate of TA100, TA1535 and TA1537 strains in the presence of metabolic activation system.
(20) Growth inhibition was not observed in TA98 and WP2uvrA (pKM101) strains in the presence of metabolic activation system, and in WP2uvrA (pKM101) strain in the absence of metabolic activation system.
(21) Therefore, the capacity of the bacterial reverse mutation assay as the present test was set as shown in Table 1 below. In addition, a negative control and a positive control were set.
(22) TABLE-US-00001 TABLE 1 strain S9 mix DOS (μg/plate) TA98 − 5,000, 2,500, 1,250, 625, 313, 156 + 5,000, 2,500, 1,250, 625, 313 TA100, TA1535, − 313, 156, 78.1, 39.1, 19.5, 9.77 TA1537 + 5,000, 2,500, 1,250, 625, 313, 156 WP2uvrA(pKM101) −/+ 5,000, 2,500, 1,250, 625, 313
(23) Precipitation of the test substance DG-EX was not observed at all doses in the presence and absence of metabolic activation system.
(24) (2) Bacterial Reverse Mutation Assay
(25) Each strain was treated with the test substance DG-EX at the above concentrations and cultured, and the number of bacterial reverse mutation colonies was visually counted. The results are shown in Table 2 below.
(26) TABLE-US-00002 TABLE 2 Dose level Individual revertant Strain Test substance (μg/plate) colony counts Mean S.D. TA98 Dimethyl sulfoxide 0 27, 26, 24 26 2 DG-EX 313 28, 25, 30 28 3 625 30, 31, 35 32 3 1,250 36, 43, 42 40 4 2,500 52, 45, 43 47 5 5,000 36, 30, 36 34 3 2-Aminoanthracene (2-AA) 1.0 358, 366, 362 362 4 TA100 Dimethyl sulfoxide 0 118, 117, 115 117 2 DG-EX 156 110, 117, 103 110 7 313 116, 116, 123 118 4 625 123, 127, 133 128 5 1,250 192, 181, 205 193 12 2,500 171, 176, 184 177 7 5,000 117*, 110*, 119* 115 5 2-Aminoanthracene (2-AA) 2.0 596, 608, 572 592 18 TA1535 Dimethyl sulfoxide 0 9, 11, 7 9 2 DG-EX 156 7, 10, 9 9 2 313 6, 8, 8 7 1 625 9, 9, 5 8 2 1,250 10, 8, 10 9 1 2,500 12, 15, 13 13 2 5,000 15*, 15*, 11* 14 2 2-Aminoanthracene (2-AA) 3.0 98, 112, 94 101 9 TA1537 Dimethyl sulfoxide 0 14, 13, 16 14 2 DG-EX 156 20, 18, 17 18 2 313 20, 18, 21 20 2 625 16, 17, 19 17 2 1,250 32, 30, 37 33 4 2,500 25*, 27*, 29* 27 2 5,000 14*, 16*, 17* 16 2 2-Aminoanthracene (2-AA) 3.0 128, 136, 122 129 7 WP2uvrA (pKM101) Dimethyl sulfoxide 0 163, 150, 151 155 7 DG-EX 313 170, 152, 169 164 10 625 170, 176, 166 171 5 1,250 180, 163, 162 168 10 2,500 176, 159, 169 168 9 5,000 168, 167, 153 163 8 2-Aminoanthracene (2-AA) 2.0 445, 462, 480 462 18 S.D: Standard Deviation *Indicates growth inhibition
(27) As a result, at the 1,250 μg/plate of the TA1537 strain in the presence of the metabolic activation system, the number of reverse mutation colonies increased more than twice as much as that of the negative control, and above 2,500 μg/plate, growth inhibition by DG-EX was observed and the number of reverse mutation colonies tended to decrease.
(28) In TA98, TA100, TA1535, TA1537 and WP2uvrA (pKM101) strains in the absence of metabolic activation system and TA98, TA100, TA1535 and WP2uvrA (pKM101) strains in the presence of metabolic activation system, regardless of the presence or absence of the metabolic activation system in the test substance group, the number of reverse mutation colonies did not exceed twice the negative control group and no dose-dependent increases were observed for all the strains of each strain. The number of reverse mutation colonies of the positive control for each strain
(29) The number of reverse mutation colonies of the positive control for each strain was more than twice as high as that of the negative control. The results are shown in
(30) As shown in
(31) 1-3 Chromosome Aberration Assay
(32) In order to confirm genotoxicity of extract of stalks and root of Daphne genkwa, a chromosome aberration assay was performed on the basis of the guidelines of Ministry of Food and Drug Safety in Biotoxtech, a GLP (Good Laboratory Practice) organization.
(33) To confirm the structural aberration of the chromosome of DG-EX obtained in the Test substance 1-1, the presence of structural aberration of the chromosome was evaluated using CHL (Chinese Hamster Lung)/IU cells, a mammalian cell line.
(34) (1) Cell Proliferation Inhibition Test
(35) For the highest capacity setting of this test, the chromosome aberration assay, cell proliferation inhibition test was carried out with the highest dose of DG-EX 5,000 μg/mL at the following doses of 2,500, 1,250, 625, 313, 156, 78.1, 39.1, and 19.5 μg/ml. The relative population doubling (RPD) after DG-EX treatment was calculated and used as an indicator of cell proliferation inhibition.
(36) As a result, cytotoxicity was observed in the presence or absence of the metabolic activation system and in the absence of the metabolic activation system of the continuous treatment method. The dose that specifically inhibited cell proliferation by more than 50% was more than 1,250 μg/mL in the presence and absence of the metabolic activation system of the short-term treatment method and more than 625 μg/mL in the absence of the metabolic activation system of the continuous treatment method.
(37) Therefore, the capacity of this test was set as shown in Table 3 below. Also, a negative control and a positive control were set.
(38) TABLE-US-00003 TABLE 3 dose of the present test treatment S9 mix (μg/mL) short-term treatment − 1,200, 600, 300, 150 + 900, 450, 225, 113 continuous treatment − 600, 300, 150, 75.0
(39) The precipitation of the test substance was observed at more than 2,500 μg/mL in the presence and absence of the metabolic activation system of the short-term treatment method and in the absence of the metabolic activation system of the continuous treatment method.
(40) (2) Chromosome Aberration Assay
(41) As a result of the chromosome aberration assay, the frequencies of cells with numerical aberration were significantly increased at 12.5% and 13.0% at 450 μg/mL and 900 μg/mL in the presence of the metabolic activation system of the short-term treatment method, which was statistically significantly increased when compared with the negative control (0%). The frequencies of cells with structural aberration were 6.0 and 8.0%, respectively, and statistically significant increase when compared with negative control (0%) (data not shown).
(42) In the absence of the metabolism activity system of the short-term treatment method and in the absence of the metabolism activity system of the continuous treatment method, the frequency of chromosome aberration was less than 5%, and no chromosomal aberration was observed, and no statistical significance was observed when compared with the negative control group.
(43) In the positive control group for each treatment group, the frequency of cells with aberrations was more than 10%, which was statistically higher than that in the negative control group.
(44) Thus, in order to clarify the positive results of the structural aberration in the presence of the metabolic activation system of the short-term treatment method, confirmation tests were carried out in the same manner as in this test with the capacity of the following Table 4.
(45) TABLE-US-00004 TABLE 4 dose of confirmation test treatment S9 mix (μg/mL) short-term treatment + 1,200, 1,100, 1,000, 900
(46) As a result of the confirmation test, the frequency of cells with numerical aberration was confirmed to be 12.5, 11.5, 10.0 and 8.5% at 900, 1,000, 1,100 and 1,200 μg/mL in the presence of the metabolic activation system of the short-term treatment method, and was statistically significantly increased when compared with the negative control group. The frequencies of cells with structural aberrations were found to be 7.5, 7.0, 8.5 and 4.5%, which was statistically significantly increased when compared to the negative control group. The results are shown in Table 5 below.
(47) TABLE-US-00005 TABLE 5 Number of cells with Number of cells with Trt-Rec No. of structural aberrations numerical aberrations Test Dose RPD S9 Time cell gap total (%) total substance (μg/mL) (%) mix (hr) analyzed ctb csb cte cse frg ctg csg gap− gap+ end pol (%) Others.sup.a) Dimethyl 0 100 + 6-18 100 1 0 0 0 0 0 0 1 (0.5) 1 (0.5) 0 0 0 (0.0) 0 sulfoxide 100 0 0 0 0 0 0 0 0 0 DG-EX 900 66.5 + 6-18 100 1 0 6 0 0 0 0 15.sup.## (7.5) 15 (7.5) 0 12 25.sup.## (12.5) 0 100 2 0 5 1 0 0 0 0 13 1,000 72.6 + 6-18 100 0 0 7 0 0 0 0 14.sup.## (7.0) 15 (7.5) 0 12 23.sup.## (11.5) 0 100 1 0 5 1 0 1 0 0 11 1,100 60.0 + 6-18 100 2 0 7 0 0 2 0 17.sup.## (8.5) 18 (9.0) 0 10 20.sup.## (10.0) 0 100 3 0 6 0 0 0 0 0 10 1,200 53.2 + 6-18 100 2 0 2 1 0 0 0 9.sup.# (4.5) 9 (4.5) 0 9 17.sup.## (8.5) 0 100 0 0 4 0 0 0 0 0 8 B[a]P 20 41.3 + 6-18 100 8 0 15 0 0 0 0 40* (20.0) 40 (20.0) 0 1 2 (1.0) 0 100 3 0 19 0 0 0 0 0 1
(48) Abbreviation: ctg: chromatid gap, csg: chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange, frg: fragmentation, end: endoreduplication, pol: polyploidy, B[a]P: Benzo[a]pyrene, RPD: Relative Population Doubling, Trt-Rec time: Treatment-Recovery times,
(49) gap−: total number of cells with structural aberration except gap,
(50) gap+: total number of cells with structural aberration including gap
(51) a): Excluded from the number of cells with chromosomal aberrations.
(52) *: p<0.01, #: p<0.05, ##: p<0.01
(53) Based on the above results, under the test conditions, the test substance DG-EX showed a frequency of cells having numerical aberration of 10% or more and a frequency of cells having a structural aberration of 5% or more and 10% or less in the presence of the metabolic activation system of the short-term treatment method, and was confirmed to have a chromosome aberration.
Example 1
Preparation of Extract of Flower of Daphne genkwa
(54) 3.5 kg of flower of Daphne genkwa was immersed in 70 L of 80% ethanol for 48 hours and filtered to separate a solid component and a first liquid component. The separated solid component were again immersed in 70 L of 80% ethanol for 24 hours and filtered to give a second liquid component. The obtained first liquid component and the second liquid component were mixed, the mixture was concentrated under reduced pressure. The residue was freeze-dried, thereby obtaining 859 g of extract of flower of Daphne genkwa (hereinafter referred to as “DGF-EX”).
Example 2
Analysis of Active Ingredients of Extract of Flower of Daphne genkwa
(55) The active ingredient of DGF-EX, an extract of flower of Daphne genkwa obtained in Example 1, was analyzed. For comparison, the active ingredient of DG-EX, the extract derived from the stem and root of Daphne genkwa obtained in Comparative Example 1-1, was also analyzed.
(56) There are two active ingredients of DGH-1 and DGH-2 in the extract derived from the stem and root of Daphne genkwa. DGH-1 has very low UV extinction coefficient and it is very difficult to analyze DGH-1 in the extract by HPLC UV method.
(57) Therefore, in order to effectively analyze both the active substances DGH-1 and DGH-2 in the extracts, they were analyzed by the multiple reaction monitoring (MRM) with LC-MS/MS. The MRM is a method of analyzing a specific substance in a complex sample by detecting a parent ion having a specific product ion.
(58) As a result, the standards DGH-1 and DGH-2 were detected at 3.81 min and 6.99 min, respectively, and detected at 1.05×10.sup.4 and 1.8×10.sup.4 intensity at 0.1 ug injection, respectively, with similar sensitivities to the MRM method (
(59) Each extract was dissolved in MeOH at a concentration of 10 μg/mL and analyzed by MRM method with LC-MS/MS after 10 μl by HPLC. The active ingredients DGH-1 and DGH-2 were detected in both the extract of flower of Daphne genkwa (DGF-EX) and the extract derived from the stem and root of Daphne genkwa (DG-EX) (
Example 3
Chromosome Aberration Assay of the Extract of Flower of Daphne genkwa
(60) In order to confirm in vitro genotoxicity of the extract of flower of Daphne genkwa obtained in Example 1, a bacterial reverse mutation assay and a chromosome aberration assay were performed as described below.
(61) 3-1 Bacterial Reverse Mutation Assay
(62) In order to confirm genotoxicity of the extract of flower of Daphne genkwa, a bacterial reverse mutation assay was conducted on the basis of the guideline of Ministry of Food and Drug Safety in Biotoxtech, a GLP (Good Laboratory Practice) organization.
(63) In order to confirm the genomic mutation induction of DGF-EX obtained in the Test 1, a bacterial reverse mutation assay in the presence of metabolic activation system (S9+) and non-existence (S9−) was performed with Salmonella typhimurium TA98, TA100, TA1535, TA1537, histidine requiring strain, and Escherichia coli WP2uvrA (pKM101), a tryptophan-requiring strain.
(64) (1) Capacity Setting Test
(65) In order to set the maximum dose of the bacterial reverse mutation assay of present test, the recommended dose of 5,000 μg/plate was used as the maximum capacity, and the capacity setting test was carried out at 1,250, 313, 78.1, and 19.5 μg/plate in common rate 4.
(66) As a result, it was found that inhibition of growth by DGF-EX was observed above 1,250 μg/plate of TA98 and TA1535 strain, above 313 μg/plate of TA100 strain, above 78.1 μg/plate of TA1537 strain in the absence of the metabolism activation system, and on 5,000 μg/plate of TA98 and TA1535 strains, and above 1,250 μg/plate of TA100 and TA1537 strains in the presence of the metabolic activation system. Growth inhibition was not observed in strain WP2uvrA (pKM101) in the presence and absence of the metabolic activation system.
(67) Therefore, the capacity of the bacterial reverse mutation assay as the present test was set as shown in Table 6 below. In addition, a negative control and a positive control were set.
(68) TABLE-US-00006 TABLE 6 dose of the present test STRAINS S9 mix (μg/plate) TA98, TA1535 − 1,250, 625, 313, 156, 78.1, 39.1 + 5,000, 2,500, 1,250, 625, 313, 156 TA100 − 313, 156, 78.1, 39.1, 19.5, 9.77 + 1,250, 625, 313, 156, 78.1, 39.1 TA1537 − 78.1, 39.1, 19.5, 9.77, 4.88, 2.44 + 1,250, 625, 313, 156, 78.1, 39.1 WP2uvrA(pKM101) −/+ 5,000, 2,500, 1,250, 625, 313
(2) Bacterial Reverse Mutation Assay
(69) Each strain was treated with the test substance DGF-EX at the above concentrations and cultured, and the number of reverse mutation colonies was visually counted. The results are shown in Table 7 below.
(70) TABLE-US-00007 TABLE 7 Dose level Individual revertant Strain Test substance (μg/plate) colony counts Mean S.D. TA98 Dimethylsulfoxide 0 24, 24, 17 25 2 DGF-EX 156 25, 27, 25 26 1 313 35, 24, 27 29 6 625 35, 30, 29 31 3 1,250 29, 22, 26 26 4 2,500 35*, 22*, 27* 28 7 5,000 22*, 25*, 22* 23 2 2-Aminoanthracene (2-AA) 1.0 436, 432, 452 440 11 TA100 Dimethylsulfoxide 0 75, 87, 85 82 6 DGF-EX 39.1 80, 87, 91 86 6 78.1 73, 87, 92 84 10 156 72, 83, 95 83 12 313 91, 87, 79 86 6 625 81, 84, 83 83 2 1,250 73*, 67*, 81* 74 7 2-Aminoanthracene (2-AA) 2.0 811, 853, 849 838 23 TA1535 Dimethylsulfoxide 0 9, 8, 10 9 1 DGF-EX 156 9, 9, 9 9 0 313 10, 8, 11 10 2 625 11, 11, 10 11 1 1,250 10, 8, 7 8 2 2,500 8*, 8*, 6* 7 1 5,000 4*, 7*, 5* 5 2 2-Aminoanthracene (2-AA) 3.0 172, 170, 151 164 12 TA1537 Dimethylsulfoxide 0 16, 11, 14 14 3 DGF-EX 39.1 15, 14, 19 16 3 78.1 14, 18, 14 15 2 156 18, 19, 15 17 2 313 20, 23, 19 21 2 625 10*, 13*, 12* 12 2 1,250 11*, 8*, 4* 8 4 2-Aminoanthracene (2-AA) 3.0 235, 210, 214 220 13 WP2uvrA(pKM101) Dimethylsulfoxide 0 161, 168, 177 169 8 DGF-EX 313 166, 172, 164 167 4 625 165, 149, 154 156 8 1,250 151, 160, 176 162 13 2,500 150, 163, 178 164 14 5,000 179, 153, 162 165 13 2-Aminoanthracene (2-AA) 2.0 549, 543, 543 545 3 S.D: Standard Deviation *indicates growth inhibition
(71) As a result, the number of reverse mutation colonies did not exceed twice the negative control group, and no dose-dependent increase was observed for all capacities of each strain, regardless of the presence or absence of metabolic activation system in the test substance group. The number of positive mutant colonies for each strain was more than twice as high as that of the negative control. The results are shown in
(72) As shown in
(73) The inhibition of growth by test material was observed above 625 μg/plate of TA98 and TA1535 strain, above 156 μg/plate of TA100 strain, on 87.1 μg/plate of TA1537 strain in the absence of the metabolism activation system, and above 2,500 μg/plate of TA98 and TA1535 strains, and on 1,250 μg/plate of TA100 and above 625 μg/plate TA1537 strains in the presence of the metabolic activation system. Growth inhibition was not observed in strain WP2uvrA (pKM101) in the presence and absence of the metabolic activation system.
(74) From the above results, under the test conditions, the test substance DGF-EX was judged not to be mutagenic.
(75) 3-2 Chromosome Aberration Assay
(76) In order to confirm genotoxicity of extract of flower of Daphne genkwa, a chromosome aberration assay was performed on the basis of the guidelines of Ministry of Food and Drug Safety in Biotoxtech, a GLP (Good Laboratory Practice) organization.
(77) To confirm the structural aberration of the chromosome of DG-EX obtained in Example 1, the presence of structural aberration of the chromosome was evaluated using CHL (Chinese Hamster Lung)/IU cells, a mammalian cell line.
(78) (1) Cell Proliferation Inhibition Test
(79) For the highest capacity setting of this test, the chromosome aberration assay, cell proliferation inhibition test was carried out with the highest dose of DGF-EX 5,000 μg/mL at the following doses of 2,500, 1,250, 625, 313, 156, 78.1, 39.1, and 19.5 μg/ml.
(80) As a result, cytotoxicity was observed in the presence or absence of the metabolic activation system and in the absence of the metabolic activation system of the continuous treatment method. The dose that specifically inhibited cell proliferation by more than 50% was more than 625 μg/mL in the presence and absence of the metabolic activation system of the short-term treatment method and more than 156 μg/mL in the absence of the metabolic activation system of the continuous treatment method. As a result of calculating the capacity to inhibit about 55% cell proliferation, 428.3 μg/mL in the absence of the metabolic activation system and 725 μg/mL in the presence of the metabolic activation system of the short-term treatment method, and 133.1 μg/mL in the absence of the metabolic activation system of the continuous treatment method.
(81) Therefore, the capacity of this test was set as shown in Table 8 below. Also, a negative control and a positive control were set.
(82) TABLE-US-00008 TABLE 8 dose of the present test treatment S9 mix (μg/mL) short-term treatment − 430, 215, 108, 53.8 + 730, 365, 183, 91.3 continuous treatment − 140, 70.0, 35, 0, 17.5
(2) Chromosome Aberration Assay
(83) As a result of the chromosome aberration assay, the frequency of cells with numerical aberration were less than 5% in the presence and absence of the metabolic activation system of the short-term treatment method and in the absence of the metabolism activity system of the continuous treatment method, and no chromosome aberration was observed, and no statistical significance was observed when compared with the negative control group. In the positive control group for each treatment group, the frequency of cells with aberrations was more than 10%, which was statistically significantly increased when compared to that in the negative control group. The results are shown in Table 9 below
(84) TABLE-US-00009 TABLE 9 Number of cells with numerical Trt-Rec No. of Number of cells with structural aberrations aberrations Test Dose RPD S9 Time cell gap total (%) total substance (μg/mL) (%) mix (hr) analyzed ctb csb cte cse frg ctg csg gap− gap+ end pol (%) Others.sup.a) Dimethyl 0 100 − 6-18 100 1 0 0 0 0 0 0 2(1.0) 2(1.0) 0 0 0(0.0) 0 sulfoxide 100 1 0 0 0 0 0 0 0 0 DGF-EX 53.8 102 − 6-18 100 not observed 100 108 102 − 6-18 100 0 0 0 0 0 0 0 0(0.0) 0(0.0) 0 0 0(0.0) 0 100 0 0 0 0 0 0 0 0 0 215 97.6 − 6-18 100 0 0 1 0 0 0 0 1(0.5) 1(0.5) 0 1 1(0.5) 0 100 0 0 0 0 0 0 0 0 0 430 75.0 − 6-18 100 0 0 1 0 0 0 0 1(0.5) 1(0.5) 0 3 5.(2.5) 0 100 0 0 0 0 0 0 0 0 2 MMC 0.1 80.4 − 6-18 100 5 0 18 0 0 0 0 38*(19.0) 38(19.0) 0 2 2(1.0) 0 100 4 0 14 0 0 0 0 0 0 Dimethyl 0 100 + 6-18 100 0 0 0 0 0 0 0 1(0.5) 1(0.5) 0 1 1(0.5) 0 sulfoxide 100 1 0 0 0 0 0 0 0 0 DGF-EX 91.3 104 + 6-18 100 not observed 100 183 98.4 + 6-18 100 0 0 0 0 0 0 0 0(0.0) 0(0.0) 0 0 0(0.0) 0 100 0 0 0 0 0 0 0 0 0 365 77.4 + 6-18 100 1 0 0 0 0 0 0 2(1.0) 2(1.0) 1 1 2(1.0) 0 100 1 0 0 0 0 0 0 0 0 730 53.4 + 6-18 100 0 0 2 0 0 0 0 2(1.0) 2(1.0) 0 3 5.(2.5) 0 100 0 0 0 0 0 0 0 0 2 B[a]P 20 57.4 + 6-18 100 5 0 15 0 0 0 0 31*(15.5) 32(16.0) 0 0 0(0.0) 0 100 2 0 13 0 0 1 0 0 0 Dimethyl 0 100 − 24-0 100 0 0 0 0 0 0 0 0(0.0) 0(0.0) 0 1 1(0.5) 0 sulfoxide 100 0 0 0 0 0 0 0 0 0 DGF-EX 17.5 97.0 − 24-0 100 not observed 100 35.0 94.4 − 24-0 100 2 0 0 0 0 0 0 2(1.0) 2(1.0) 0 1 1(0.5) 0 100 0 0 0 0 0 0 0 0 0 70.0 93.5 − 24-0 100 0 0 0 0 0 0 0 1(0.5) 1(0.5) 0 1 1(0.5) 0 100 0 1 0 0 0 0 0 0 0 140 57.1 − 24-0 100 0 0 0 0 0 0 0 1(0.5) 1(0.5) 0 1 1(0.5) 0 100 0 1 0 0 0 1 0 0 0 MMC 0.1 66.0 − 24-0 100 6 0 25 0 0 0 0 52*(26.0) 52(26.0) 0 0 0(0.0) 0 100 6 1 20 1 0 0 0 0 0
(85) Abbreviation: ctg: chromatid gap, csg: chromosome gap, ctb: chromatid break, cte:chromatid exchange, csb: chromosome break, cse: chromosome exchange, frg: fragmentation, end: endoreduplication, pol: polyploidy, MMC: Mitomycin C, B[a]P: Benzo[a]pyrene, RPD: Relative Population Doubling, Trt-Rec time: Treatment-Recovery times, gap−: total number of cells with structural aberration except gap, gap+: total number of cells with structural aberration including gap
(86) a): Excluded from the number of cells with chromosomal aberrations
(87) *: p<0.01
(88) From the above results, the chromosomal abnormality of the test substance DGF-EX was judged to be negative under this test condition.
Example 4
Confirming the Effect of the Extract of Flower of Daphne genkwa on the Nurr1 Activity
(89) To confirm the Nurr1 activation effect of DGF-EX obtained in Example 1, Nurr1 activity experiments were performed using the GAL4 assay system.
(90) After a GAL4-LBD plasmid, a luciferase plasmid to which GAL4 can bind, and β-galactosidase were transfected into BE(2)C cells, which are neuroblasts of human origin, DGF-EX was treated for 16 hours at concentrations of 1, 25 and 40 ppm. The cells thus treated were cultured in a 5% carbon dioxide incubator at 37° C. for 20 hours, and the extract was dissolved in DMSO and treated. The control group was treated with 1% DMSO and then subjected to luciferase fluorescence analysis. The results are shown in
(91) As shown in
(92) Also, the Nurr1 activity of DGF-EX and DG-EX obtained in Comparative Example 1 was compared in the same manner as described above, and the results are shown in
(93) As shown in
Example 5
Identification of the Effect of the Extract of Flower of Daphne genkwa in Animal Model of Parkinson's Disease
(94) This study was carried out to investigate the effect of the extract of flower of Daphne genkwa in animal models of Parkinson's disease.
(95) In order to specifically kill dopaminergic neurons in substantia nigra of midbrain, 6-OHDA(0.2 μg/μl, final volume 5 μl) was directly injected into the AP(−4.3), ML(−1.8), DV(−8.2), and AP(−5.0), ML(−1.8), DV(−8.2) regions of the brain using a stereotaxic tool in 18 6-week-old SD rats (Coatec.) to produce an animal model of Parkinson's disease as experimental group. Desipramine was administered at a dose of 25 mg/kg 30 minutes before the administration of 6-OHDA to inhibit cell death other than dopaminergic neurons. As a control group, 6 6-week-old SD rats (Coatec.) were injected with PBS instead of 6-OHDA.
(96) Six of the 6-OHDA-lesioned 18 rats were vehicle treated (control group), and another 6 rats were fed a diet supplemented with 50 mg/kg of the daily dose of the extract of flower of Daphne genkwa, and the remaining 6 rats were fed a diet supplemented with 100 mg/kg of the daily dose of the extract of flower of Daphne genkwa.
(97) A rotation test was performed at two and 4 weeks after surgery. When the dopamine receptor agonist, apomorphine is injected into an animal model of Parkinson's disease, a denervation supersensitivity of dopamine induces rotation to the opposite side of the lesion, which can be measured quantitatively using a rotometer. The results are shown in
(98) As shown in
Example 6
Immunohistochemical Analysis of the Effect of the Extract of Flower of Daphne genkwa in Animal Model of Parkinson's Disease
(99) In Example 5, the therapeutic effect in the animal model of Parkinson's disease of the extract of flower of Daphne genkwa was confirmed, and we further investigated whether the death of dopaminergic neurons in the substantia nigra of midbrain was actually inhibited by the administration of the extract of flower of Daphne genkwa.
(100) An animal model of Parkinson's disease was prepared in the same manner as in Example 5, and rats were sacrificed at 4 weeks after the surgery, and followed by perfusion with 4% paraformaldehyde. Brains were removed, and cut into a 40 μm coronal section using a vibratome and then subjected to immunostaining with a tyrosine hydroxylase antigen. The sections were treated with 3% hydrogen peroxide solution and washed 3 times with PBS. Then, after the sections were blocked with 5% horse serum for 1 hour at room temperature, tyrosine hydroxylase antigen was treated overnight at 4° C. The sections were washed with PBS and then treated with secondary antirabbit IgG and treated with avidin-biotinylated peroxidase complex and 3,3′-diaminobenzidine.
(101) The color-developed and dyed dopaminergic neurons were observed under a microscope. The results are shown in
(102) As shown in
(103) Furthermore, as shown in
(104) This indicates that the extract of flower of Daphne genkwa (DGF) strongly inhibited dopaminergic neurons death by 6-OHDA.