KR20240160833A - Composition for preventing, ameliorating or treating Parkinson's disease comprising jellyfish venom as effective component - Google Patents
Composition for preventing, ameliorating or treating Parkinson's disease comprising jellyfish venom as effective component Download PDFInfo
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- KR20240160833A KR20240160833A KR1020230057640A KR20230057640A KR20240160833A KR 20240160833 A KR20240160833 A KR 20240160833A KR 1020230057640 A KR1020230057640 A KR 1020230057640A KR 20230057640 A KR20230057640 A KR 20230057640A KR 20240160833 A KR20240160833 A KR 20240160833A
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- disease
- parkinson
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- A—HUMAN NECESSITIES
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
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- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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Abstract
본 발명은 해파리 독소를 유효성분으로 함유하는 파킨슨병의 예방, 개선 또는 치료용 조성물에 관한 것으로, 해파리 독소, 구체적으로 작은상자 해파리(Carybdea brevipedalia)로부터 획득한 해파리 독소가 1-메틸-4-페닐피리디늄 아이오다이드(1-methyl-4-phenylpyridinium iodide)에 의해 촉진된 신경세포의 사멸을 억제하고, 파킨슨이 유발된 동물모델에서, 감소된 운동능력을 향상시키며, 파킨슨이 유발된 동물모델의 뇌 조직에서 도파민 전구체 관련 효소인 TH(Tyrosine hydroxylase)의 발현을 증가시키는 효과가 우수하므로, 파킨슨병의 예방, 개선 또는 치료용 조성물로 유용하게 사용될 수 있다. The present invention relates to a composition for preventing, improving or treating Parkinson's disease containing jellyfish venom as an active ingredient. Since jellyfish venom, specifically jellyfish venom obtained from the small box jellyfish ( Carybdea brevipedalia ), has an excellent effect of inhibiting neuronal cell death promoted by 1-methyl-4-phenylpyridinium iodide, improving reduced motor ability in an animal model induced with Parkinson's disease, and increasing the expression of TH (Tyrosine hydroxylase), an enzyme related to a dopamine precursor, in the brain tissue of an animal model induced with Parkinson's disease, the composition can be usefully used as a composition for preventing, improving or treating Parkinson's disease.
Description
본 발명은 해파리 독소를 유효성분으로 함유하는 파킨슨병의 예방, 개선 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for preventing, improving or treating Parkinson's disease containing jellyfish toxin as an active ingredient.
파킨슨병(parkinson's disease; PD)은 알츠하이머 치매와 더불어 대표적인 퇴행성 뇌신경질환의 하나로서, 도파민 호르몬을 분비하는 뇌의 흑질부위 신경세포가 점점 사멸해가면서 발병한다. 도파민은 뇌의 기저핵에 작용하여 우리가 원하는 대로 몸을 정교하게 움직일 수 있도록 하는 중요한 신경 전달계 물질인데, 이를 분비하는 신경세포들이 사멸하면서 떨림증(tremor), 경직(rigidity), 운동완서(bradykinesia), 자세의 불안정(postural instability) 등의 운동성 장애가 생긴다.Parkinson's disease (PD) is one of the representative degenerative brain diseases along with Alzheimer's disease. It is caused by the gradual death of nerve cells in the substantia nigra of the brain that secrete dopamine hormone. Dopamine is an important neurotransmitter that acts on the basal ganglia of the brain and allows us to move our bodies precisely as we wish. When nerve cells that secrete dopamine die, movement disorders such as tremors, rigidity, bradykinesia, and postural instability occur.
파킨슨병은 65세 인구에서 약 1% 정도가 발병하며 나이가 들수록 그 발병률이 증가하는 것으로 알려져 있으나, 정확한 원인을 알 수 없는 특발성(idiopathic)인 경우가 대부분이며, 일부 유전적 요인, 환경적 요인이 알려져 있다. 주요 병인으로는, 중뇌 흑질의 도파민 신경세포 사멸, 알파-시누클린(α-synuclein) 단백질 응집, 신경염증 등이 있다.Parkinson's disease occurs in about 1% of the population over 65 years of age, and its incidence is known to increase with age. However, most cases are idiopathic, with no known exact cause, and some genetic and environmental factors are known. The main causes include dopaminergic neuron death in the substantia nigra, alpha-synuclein protein aggregation, and neuroinflammation.
현재 초기 파킨슨병의 증상을 완화시키는 약물로는 레보도파(Levodopa, L-DOPA), 도파민 작용제(agonist), 항콜린 제제, 아만타딤 등이 있으나, 이들 약물은 지속적인 도파민 신경의 손실을 막아내지 못하므로 5~6년 내에는 약물 효과가 급격히 감소하며, 이후에는 이상운동증, 불면, 식욕감소 등의 부작용이 증가한다는 문제가 있다. 따라서, 파킨슨병을 치료할 수 있는 새로운 약물의 개발이 절실한 실정이다.Currently, medications that alleviate the symptoms of early Parkinson's disease include levodopa (L-DOPA), dopamine agonists, anticholinergics, and amantadim. However, these drugs do not prevent the continuous loss of dopamine neurons, so the drug effect decreases rapidly within 5 to 6 years, and thereafter, side effects such as dyskinesia, insomnia, and decreased appetite increase. Therefore, the development of new drugs that can treat Parkinson's disease is urgently needed.
한편, 해양식물, 해양동물 및 해양미생물로부터 생리활성물질을 분리하거나 그들의 시스템 공정과 기능을 이용하고자 하는 학문 또는 산업을 마린 바이오프로스펙팅(marine bioprospecting)이라고 한다. 자포동물문(Cnidaria)은 해파리, 산호, 말미잘 등이 포함되어 있는 동물군으로 11,000여 종 이상으로 구성되어 있으며 이들로부터 분리한 생리활성물질이 현재까지 3,000여 종 이상이다. 그 중 해파리는 지구 온난화 등으로 인해 전 세계적으로 출현빈도가 잦으며, 이들의 대량출현으로 어업(어망손상 및 어획량 감소), 발전소, 사람(해파리 독소)에게 많은 피해를 주고 있다. Meanwhile, the academic discipline or industry that separates physiologically active substances from marine plants, marine animals, and marine microorganisms or utilizes their system processes and functions is called marine bioprospecting. Cnidaria is an animal group that includes jellyfish, corals, and sea anemones, and consists of over 11,000 species, and more than 3,000 physiologically active substances have been separated from them to date. Among them, jellyfish appear frequently around the world due to global warming, etc., and their mass appearance causes a lot of damage to the fisheries (damage to fishing nets and decrease in catch), power plants, and people (jellyfish toxin).
지금까지 해파리 독소의 약리 독성학적 작용은 별로 알려진 바가 없었으며, 최근에 그 작용 기작의 일부가 보고되고 있을 뿐이다. 일반적으로 독소를 가지는 동물에 대한 연구는 뱀, 전갈, 거미, 벌 등의 육상동물에 치우쳐져 있었다. 이에 반해, 해파리 독소에 대한 연구는 심순환기에 초점이 맞추어져 있고, 실제로 해파리 독소는 심장 및 순환기에 강한 억제작용을 나타내고 있다. Until now, the pharmacological toxicological effects of jellyfish toxins have not been well known, and only recently have some of their mechanisms of action been reported. In general, research on animals with toxins has been focused on terrestrial animals such as snakes, scorpions, spiders, and bees. In contrast, research on jellyfish toxins has focused on the cardiovascular system, and jellyfish toxins actually show strong inhibitory effects on the heart and circulatory system.
해파리는 종류에 따라 독소의 종류가 달라 각기 다른 양상의 약리학적 작용을 나타내고 있지만, 아직 구체적으로 밝혀진 바는 없으므로, 해파리 독소를 이용하여 해파리를 해로운 동물이 아닌 마린 프로스펙팅의 자원으로 이용하고자 한다.Jellyfish have different types of toxins depending on their species, and each exhibits different pharmacological effects, but this has not been specifically identified yet. Therefore, we aim to utilize jellyfish toxins as a resource for marine prospecting rather than as a harmful animal.
한편, 한국공개특허 제2009-0114999호에는 봉독을 포함하는 간경변 치료용 약학조성물에 대해 개시하고 있으며, 한국등록특허 제1536215호에는 해파리 콜라겐 가수분해물을 유효성분으로 함유하는 항산화, 항염, 미백 및 주름 개선용 화장료 조성물에 대해 개시하고 있지만, 본 발명의 해파리 독소를 유효성분으로 함유하는 파킨슨병의 예방, 개선 또는 치료용 조성물에 대해서는 아직까지 개시된 바 없다.Meanwhile, Korean Patent Publication No. 2009-0114999 discloses a pharmaceutical composition for treating liver cirrhosis containing bee venom, and Korean Registration Patent No. 1536215 discloses a cosmetic composition for antioxidation, anti-inflammation, whitening, and wrinkle improvement containing jellyfish collagen hydrolysate as an active ingredient. However, a composition for preventing, improving, or treating Parkinson's disease containing the jellyfish toxin of the present invention as an active ingredient has not yet been disclosed.
본 발명은 상기와 같은 요구에 의해 도출된 것으로, 해파리 독소를 유효성분으로 함유하는 파킨슨병의 예방, 개선 또는 치료용 조성물을 제공하고, 해파리 독소, 구체적으로 작은상자 해파리(Carybdea brevipedalia)로부터 획득한 해파리 독소가 신경세포의 사멸을 억제하고, 파킨슨이 유발된 동물모델에서, 감소된 운동능력을 향상시키며, 파킨슨이 유발된 동물모델의 뇌 조직에서 도파민 전구체 관련 효소인 TH(Tyrosine hydroxylase)의 발현을 증가시키는 것을 확인함으로써, 본 발명을 완성하였다.The present invention was derived from the above needs, and provides a composition for preventing, improving or treating Parkinson's disease containing jellyfish toxin as an active ingredient, and completed the present invention by confirming that jellyfish toxin, specifically jellyfish toxin obtained from the small box jellyfish ( Carybdea brevipedalia ), inhibits the death of nerve cells, improves reduced motor ability in an animal model induced with Parkinson's disease, and increases the expression of TH (Tyrosine hydroxylase), an enzyme related to a dopamine precursor, in the brain tissue of an animal model induced with Parkinson's disease.
상기 과제를 해결하기 위하여, 본 발명은 해파리 독소를 유효성분으로 함유하는 파킨슨병의 예방 또는 치료용 약학 조성물을 제공한다. To solve the above problem, the present invention provides a pharmaceutical composition for preventing or treating Parkinson's disease containing jellyfish toxin as an active ingredient.
또한, 본 발명은 해파리 독소를 유효성분으로 함유하는 파킨슨병의 예방 또는 개선용 건강기능식품 조성물을 제공한다. In addition, the present invention provides a health functional food composition for preventing or improving Parkinson's disease containing jellyfish toxin as an effective ingredient.
또한, 본 발명은 해파리 독소를 유효성분으로 함유하는 파킨슨병의 예방 또는 치료용 수의학적 조성물을 제공한다.In addition, the present invention provides a veterinary composition for preventing or treating Parkinson's disease containing jellyfish toxin as an active ingredient.
또한, 본 발명은 해파리 독소를 유효성분으로 함유하는 파킨슨병의 예방 또는 개선용 사료 첨가제를 제공한다.In addition, the present invention provides a feed additive for preventing or improving Parkinson's disease containing jellyfish toxin as an effective ingredient.
본 발명은 해파리 독소를 유효성분으로 함유하는 파킨슨병의 예방, 개선 또는 치료용 조성물에 관한 것으로, 해파리 독소, 구체적으로 작은상자 해파리(Carybdea brevipedalia)로부터 획득한 독소가 신경세포의 사멸을 억제하고, 도파민 전구체 관련 효소인 TH(Tyrosine hydroxylase)의 발현을 증가시키는 효과가 우수하고, 파킨슨이 유발된 동물모델에서, 막대실험, 악력 시험 및 좁은 빔 실험(time on narrow beam test)을 통해 감소된 운동능력을 향상시키는 효과가 우수한 것을 확인하였으며, 동물모델의 뇌 조직에서 해파리 독소 처리에 의해 도파민 전구체 관련 효소인 TH(Tyrosine hydroxylase)의 발현이 현저히 증가되는 효과가 있다. The present invention relates to a composition for preventing, improving or treating Parkinson's disease containing jellyfish toxin as an active ingredient. The jellyfish toxin, specifically, the toxin obtained from the small box jellyfish ( Carybdea brevipedalia ), has an excellent effect of inhibiting the death of nerve cells and increasing the expression of TH (Tyrosine hydroxylase), an enzyme related to a dopamine precursor. In an animal model induced with Parkinson's disease, it was confirmed to have an excellent effect of improving reduced motor ability through a rod test, a grip strength test and a time on narrow beam test. In the brain tissue of an animal model, the expression of TH (Tyrosine hydroxylase), an enzyme related to a dopamine precursor, is significantly increased by treatment with jellyfish toxin.
도 1은 신경모세포종(human neuroblastoma) 유래 세포주인 SH-SY5Y에서, 해파리 독소(Carybdea brevipedalia venom, CbV; A) 및 MPP+(1-methyl-4-phenylpyridinium iodide; B)의 세포독성을 확인한 결과이다.
도 2는 SH-SY5Y에 MPP+를 처리하여 유도된 신경 독성모델에서, 해파리 독소(CbV) 처리에 따른 세포생존율 변화를 확인(A)하고, 세포를 관찰한 결과(B)이다. **은 무처리군 대비 MPP+ 단독 처리군의 세포생존율이 통계적으로 유의미하게 감소하였다는 것을 의미하며, p<0.01이다. #, ##은 MPP+ 단독 처리군 대비 CbV를 함께 처리한 군의 세포생존율이 통계적으로 유의미하게 증가하였다는 것을 의미하며, #은 p<0.05, ##은 p<0.01이다.
도 3은 SH-SY5Y에 MPP+를 처리하여 유도된 신경 독성모델에서, 해파리 독소(CbV) 처리에 따른 세포 형태를 DAPI 염색을 통해 확인(A)하고, 세포자살이 일어난 세포수를 정량한 결과(B)이다. **은 무처리군 대비 MPP+ 단독 처리군의 세포자살이 일어난 세포수가 통계적으로 유의미하게 증가하였다는 것을 의미하며, p<0.01이다. #, ##은 MPP+ 단독 처리군 대비 CbV를 함께 처리한 군의 세포자살이 일어난 세포수가 통계적으로 유의미하게 감소하였다는 것을 의미하며, #은 p<0.05, ##은 p<0.01이다.
도 4는 SH-SY5Y에 MPP+를 처리하여 유도된 신경 독성모델에서, 해파리 독소(CbV) 처리에 따른 TH(Tyrosine hydroxylase) 단백질 발현 변화를 웨스턴 블롯(Western blot)으로 확인한 결과이다.
도 5는 파킨슨병 동물모델을 이용한 실험 일정을 도식화한 것이다.
도 6은 MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) 투여를 통해 유도된 파킨슨병 동물모델에서, 해파리 독소가 투여된 마우스가 막대를 내려오기 위해 아래로 향하는 시간 변화를 확인한 막대 실험(Pole test) 결과이다. con은 아무것도 처리하지 않은 정상 대조군이고, MPTP는 MPTP를 투여하여 파킨슨병을 유도한 군이고, MPTP+CbV 25는 MPTP 및 25㎍/kg의 CbV를 투여한 군이고, MPTP+CbV 50은 MPTP 및 50㎍/kg의 CbV를 투여한 군이며, MPTP+L-Dopa는 MPTP 및 레보도파(L-Dopa)를 투여한 양성 대조군이다. **은 정상군 대비 파킨슨병 동물모델(MPTP)에서 막대를 내려오기 위해 아래로 향하는 시간이 통계적으로 유의미하게 증가하였다는 것을 의미하며, p<0.01이다. #, ##은 파킨슨병 동물모델 대비 파킨슨병 동물모델에 해파리 독소(CbV) 또는 L-Dopa를 투여한 군의 막대를 내려오기 위해 아래로 향하는 시간이 통계적으로 유의미하게 감소하였다는 것을 의미하며, #은 p<0.05, ##은 p<0.01이다.
도 7은 MPTP 투여를 통해 유도된 파킨슨병 동물모델에서, 해파리 독소가 투여된 마우스의 악력 강도 변화를 확인한 악력 시험(Grip strength) 결과이다. 그립 강도는 그램(g) 단위로 나타냈다. con은 아무것도 처리하지 않은 정상 대조군이고, MPTP는 MPTP를 투여하여 파킨슨병을 유도한 군이고, MPTP+CbV 25는 MPTP 및 25㎍/kg의 CbV를 투여한 군이고, MPTP+CbV 50은 MPTP 및 50㎍/kg의 CbV를 투여한 군이며, MPTP+L-Dopa는 MPTP 및 레보도파(L-Dopa)를 투여한 양성 대조군이다. **은 정상군 대비 파킨슨병 동물모델(MPTP)의 악력이 통계적으로 유의미하게 감소하였다는 것을 의미하며, p<0.01이다. #, ##은 파킨슨병 동물모델 대비 파킨슨병 동물모델에 해파리 독소(CbV) 또는 L-Dopa를 투여한 군의 악력이 통계적으로 유의미하게 증가하였다는 것을 의미하며, #은 p<0.05, ##은 p<0.01이다.
도 8은 MPTP 투여를 통해 유도된 파킨슨병 동물모델에서, 해파리 독소가 투여된 마우스의 모터 조정 능력을 확인한 좁은 빔 실험(time on narrow beam test) 결과이다. 마우스를 바닥 위 100cm 높이에 위치한 고정식 목재의 좁은 평면 빔(1cm)에서 움직이도록 훈련한 후, 각 마우스가 한쪽 끝에서 다른 쪽 끝으로 걷는 시간을 기록하였다. con은 아무것도 처리하지 않은 정상 대조군이고, MPTP는 MPTP를 투여하여 파킨슨병을 유도한 군이고, MPTP+CbV 25는 MPTP 및 25㎍/kg의 CbV를 투여한 군이고, MPTP+CbV 50은 MPTP 및 50㎍/kg의 CbV를 투여한 군이며, MPTP+L-Dopa는 MPTP 및 레보도파(L-Dopa)를 투여한 양성 대조군이다. *은 정상군 대비 파킨슨병 동물모델(MPTP)의 한쪽 끝에서 다른 쪽 끝으로 걷는 시간이 통계적으로 유의미하게 증가하였다는 것을 의미하며, p<0.05이다. #은 파킨슨병 동물모델 대비 파킨슨병 동물모델에 해파리 독소(CbV) 또는 L-Dopa를 투여한 군의 한쪽 끝에서 다른 쪽 끝으로 걷는 시간이 통계적으로 유의미하게 감소하였다는 것을 의미하며, p<0.05이다.
도 9는 MPTP 투여를 통해 유도된 파킨슨병 동물모델의 뇌 조직에서, 해파리 독소(CbV) 투여에 따른 TH(Tyrosine hydroxylase) 발현 변화를 면역화학염색으로 확인한 결과이다. con은 아무것도 처리하지 않은 정상 대조군이고, MPTP는 MPTP를 투여하여 파킨슨병을 유도한 군이고, MPTP+CbV 25는 MPTP 및 25㎍/kg의 CbV를 투여한 군이고, MPTP+CbV 50은 MPTP 및 50㎍/kg의 CbV를 투여한 군이며, MPTP+L-Dopa는 MPTP 및 레보도파(L-Dopa)를 투여한 양성 대조군이다.
도 10은 MPTP 투여를 통해 유도된 파킨슨병 동물모델의 뇌 선조체에서, 해파리 독소(CbV) 투여에 따른 TH(Tyrosine hydroxylase) 단백질 발현 변화를 확인한 결과이다. L-Dopa는 양성 대조군으로 사용하였다. Figure 1 shows the results of confirming the cytotoxicity of jellyfish toxin ( Carybdea brevipedalia venom, CbV; A) and MPP+ (1-methyl-4-phenylpyridinium iodide; B) in SH-SY5Y, a cell line derived from human neuroblastoma.
Figure 2 shows the results of confirming the change in cell viability according to jellyfish toxin (CbV) treatment in the neurotoxicity model induced by treating SH-SY5Y with MPP+ (A) and observing the cells (B). ** indicates that the cell viability of the MPP+ only treatment group statistically significantly decreased compared to the untreated group, and p<0.01. #, ## indicate that the cell viability of the group treated with CbV statistically significantly increased compared to the MPP+ only treatment group, and # indicates p<0.05, and ## indicates p<0.01.
Figure 3 shows the results of confirming the cell morphology according to jellyfish toxin (CbV) treatment through DAPI staining (A) and quantifying the number of cells in which apoptosis occurred (B) in a neurotoxicity model induced by treating SH-SY5Y with MPP+. ** indicates a statistically significant increase in the number of cells in which apoptosis occurred in the MPP+ only treatment group compared to the untreated group, and p<0.01. #, ## indicate a statistically significant decrease in the number of cells in which apoptosis occurred in the group treated with CbV together compared to the MPP+ only treatment group, and # indicates p<0.05, and ## indicates p<0.01.
Figure 4 shows the results of Western blot analysis of changes in TH (Tyrosine hydroxylase) protein expression following jellyfish toxin (CbV) treatment in a neurotoxicity model induced by treating SH-SY5Y with MPP+.
Figure 5 is a schematic diagram of an experimental schedule using an animal model of Parkinson's disease.
Figure 6 shows the results of the pole test in which the time taken by mice administered jellyfish toxin to descend a pole is confirmed in a Parkinson's disease animal model induced by administration of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). con is a normal control group that was not treated with anything, MPTP is a group in which Parkinson's disease was induced by administration of MPTP, MPTP+CbV 25 is a group administered MPTP and 25 μg/kg of CbV, MPTP+CbV 50 is a group administered MPTP and 50 μg/kg of CbV, and MPTP+L-Dopa is a positive control group administered MPTP and levodopa (L-Dopa). ** means that the time taken to descend the pole was statistically significantly increased in the Parkinson's disease animal model (MPTP) compared to the normal group, and p<0.01. #, ## indicate that the time taken to descend the rod was statistically significantly reduced in the group administered jellyfish venom (CbV) or L-Dopa to the Parkinson's disease animal model compared to the Parkinson's disease animal model, and # indicates p<0.05, and ## indicates p<0.01.
Figure 7 shows the results of a grip strength test that confirmed the change in grip strength of mice administered jellyfish toxin in a Parkinson's disease animal model induced by MPTP administration. Grip strength was expressed in units of grams (g). con is a normal control group that was not treated with anything, MPTP is a group in which Parkinson's disease was induced by MPTP administration, MPTP+CbV 25 is a group administered MPTP and 25 μg/kg of CbV, MPTP+CbV 50 is a group administered MPTP and 50 μg/kg of CbV, and MPTP+L-Dopa is a positive control group administered MPTP and levodopa (L-Dopa). ** means that the grip strength of the Parkinson's disease animal model (MPTP) was statistically significantly reduced compared to the normal group, and p<0.01. #, ## indicate that the grip strength of the group administered jellyfish venom (CbV) or L-Dopa to the Parkinson's disease animal model was statistically significantly increased compared to the Parkinson's disease animal model, and # indicates p<0.05, and ## indicates p<0.01.
Figure 8 shows the results of a time on narrow beam test to confirm the motor coordination ability of mice administered jellyfish toxin in an animal model of Parkinson's disease induced by MPTP administration. After training the mice to move on a narrow flat beam (1 cm) of a fixed wooden structure placed 100 cm above the floor, the time taken for each mouse to walk from one end to the other was recorded. con is a normal control group that was not treated with anything, MPTP is a group in which Parkinson's disease was induced by administering MPTP, MPTP+CbV 25 is a group administered MPTP and 25 μg/kg of CbV, MPTP+CbV 50 is a group administered MPTP and 50 μg/kg of CbV, and MPTP+L-Dopa is a positive control group administered MPTP and levodopa (L-Dopa). * indicates that the walking time from one end to the other of the Parkinson's disease animal model (MPTP) increased statistically significantly compared to the normal group, and p<0.05. # indicates that the walking time from one end to the other of the group administered jellyfish toxin (CbV) or L-Dopa to the Parkinson's disease animal model statistically significantly decreased compared to the Parkinson's disease animal model, and p<0.05.
Figure 9 shows the results of immunochemical staining for changes in TH (Tyrosine hydroxylase) expression in the brain tissue of an animal model of Parkinson's disease induced by MPTP administration according to jellyfish toxin (CbV) administration. con is a normal control group that was not treated with anything, MPTP is a group in which Parkinson's disease was induced by MPTP administration, MPTP+CbV 25 is a group that was administered MPTP and 25㎍/kg of CbV, MPTP+CbV 50 is a group that was administered MPTP and 50㎍/kg of CbV, and MPTP+L-Dopa is a positive control group that was administered MPTP and levodopa (L-Dopa).
Figure 10 shows the results of confirming the change in TH (Tyrosine hydroxylase) protein expression in the brain striatum of an animal model of Parkinson's disease induced by MPTP administration following jellyfish venom (CbV) administration. L-Dopa was used as a positive control.
본 발명의 목적을 달성하기 위하여, 본 발명은 해파리 독소를 유효성분으로 함유하는 파킨슨병의 예방 또는 치료용 약학 조성물을 제공한다. In order to achieve the purpose of the present invention, the present invention provides a pharmaceutical composition for preventing or treating Parkinson's disease containing jellyfish toxin as an active ingredient.
상기 해파리는 작은상자 해파리(Carybdea brevipedalia)인 것이지만, 이에 한정하는 것은 아니다. The jellyfish mentioned above is, but is not limited to, the small box jellyfish ( Carybdea brevipedalia ).
본 발명의 일 구현 예에서, 상기 해파리 독소는 In one embodiment of the present invention, the jellyfish toxin is
1) 해파리로부터 분리된 촉수를 세척한 후, 촉수에 바닷물을 첨가한 다음 자가분해하는 단계;1) A step of washing the tentacles separated from the jellyfish, adding seawater to the tentacles, and then autolyzing them;
2) 상기 단계 1) 이후에, 상기 단계 1)의 자가분해물을 원심분리하여 자포가 포함된 상등액을 수집하고, 원심분리 후 남은 침전물에 다시 바닷물을 첨가한 후 자가분해한 다음 원심분리하여 상등액을 수집하는 과정을 1~4회 반복하는 단계;2) After the above step 1), the autolysate of the above step 1) is centrifuged to collect the supernatant containing the spores, and the process of adding seawater to the sediment remaining after centrifugation, autolyzing, and then centrifuging to collect the supernatant is repeated 1 to 4 times;
3) 상기 단계 2)에서 획득된 상등액을 동결건조하여 자포 분말을 획득하는 단계;3) A step of freeze-drying the supernatant obtained in step 2) to obtain a powder of spores;
4) 상기 단계 3)에서 획득한 자포 분말에 인산완충식염수 및 유리구슬을 첨가한 후, 진탕하여 물리적인 충격을 가한 다음 얼음을 이용해 냉각시키는 과정을 8~12회 반복하여 독소를 추출하는 단계; 및4) A step of extracting the toxin by adding phosphate buffered saline and glass beads to the powder of the nematode obtained in step 3), shaking to apply physical impact, and then cooling using ice, repeating the process 8 to 12 times; and
5) 상기 단계 4) 이후에, 원심분리한 다음 상층액의 독소를 획득하는 단계;를 포함하여 제조될 수 있고, 5) After the above step 4), a step of centrifuging and then obtaining the toxin of the supernatant can be manufactured, including;
바람직하게는 Preferably
1) 해파리로부터 분리된 촉수를 바닷물로 세척한 후, 촉수 1 중량부에 대하여 2~4 중량부의 바닷물을 첨가한 다음 20~28시간 동안 자가분해하는 단계;1) A step of washing tentacles separated from a jellyfish with seawater, adding 2 to 4 parts by weight of seawater to 1 part by weight of the tentacles, and then autolyzing for 20 to 28 hours;
2) 상기 단계 1) 이후에, 상기 단계 1)의 자가분해물을 원심분리하여 자포가 포함된 상등액을 수집하고, 원심분리 후 남은 침전물에 다시 바닷물을 첨가한 후 20~28시간 동안 자가분해한 다음 원심분리하여 상등액을 수집하는 과정을 1~4회 반복하는 단계;2) After the above step 1), the autolysate of the above step 1) is centrifuged to collect the supernatant containing the spores, seawater is added to the sediment remaining after the centrifugation, and the process of autolyzing for 20 to 28 hours and then centrifuging to collect the supernatant is repeated 1 to 4 times;
3) 상기 단계 2)에서 획득한 상등액을 동결건조하여 자포 분말을 획득하는 단계;3) A step of freeze-drying the supernatant obtained in step 2) to obtain a powder of spores;
4) 상기 단계 3)에서 획득한 자포 분말 70mg에 인산완충식염수 0.8~1.2mL 및 유리구슬을 첨가한 후, 진탕하여 물리적인 충격을 가한 다음 얼음을 이용해 1분간 냉각시키는 과정을 8~12회 반복하여 독소를 추출하는 단계; 및4) A step of extracting the toxin by adding 0.8 to 1.2 mL of phosphate buffered saline and glass beads to 70 mg of the nematode powder obtained in the above step 3), shaking to apply physical impact, and then cooling with ice for 1 minute, repeating the process 8 to 12 times; and
5) 상기 단계 4) 이후에, 원심분리한 다음 상층액의 독소를 획득하는 단계;를 포함하여 제조될 수 있으며, 5) After the above step 4), a step of centrifuging and then obtaining the toxin in the supernatant can be manufactured, including;
더 바람직하게는 More preferably
1) 해파리로부터 분리된 촉수를 바닷물로 세척한 후, 촉수 1 중량부에 대하여 3 중량부의 바닷물을 첨가한 후, 24시간 동안 자가분해하는 단계;1) A step of washing tentacles separated from a jellyfish with seawater, adding 3 parts by weight of seawater to 1 part by weight of the tentacles, and then autolyzing for 24 hours;
2) 상기 단계 1) 이후에, 상기 단계 1)의 자가분해물을 원심분리(1,000×g, 10분)하여 자포가 포함된 상등액을 수집하고, 원심분리 후 남은 침전물에 다시 바닷물을 첨가한 후 24시간 동안 자가분해한 다음 원심분리(1,000×g, 10분)하여 상등액을 수집하는 과정을 3~4회 반복하는 단계;2) After the above step 1), the autolysate of the above step 1) is centrifuged (1,000×g, 10 minutes) to collect the supernatant containing the spores, seawater is added to the sediment remaining after the centrifugation, autolysed for 24 hours, and then centrifuged (1,000×g, 10 minutes) to collect the supernatant. This process is repeated 3 to 4 times;
3) 상기 단계 2)에서 획득한 상등액을 동결건조하여 자포 분말을 획득하는 단계;3) A step of freeze-drying the supernatant obtained in step 2) to obtain a powder of spores;
4) 상기 단계 3)에서 획득한 자포 분말 70mg에 인산완충식염수 1mL 및 유리구슬을 첨가한 후, 3000rpm으로 40초 동안 진탕하여 물리적인 충격을 가한 다음 얼음을 이용해 1분간 냉각시키는 과정을 10회 반복하여 독소를 추출하는 단계; 및4) A step of extracting toxin by adding 1 mL of phosphate buffered saline and glass beads to 70 mg of the nematode powder obtained in the above step 3), shaking at 3000 rpm for 40 seconds to apply physical impact, and then cooling with ice for 1 minute, repeating the process 10 times; and
5) 상기 단계 4) 이후에, 원심분리(12000×g, 30분)한 다음 상층액의 독소를 획득하는 단계;를 포함하여 제조되는 것이지만, 이에 제한되는 것은 아니다. 5) A method for producing a toxin, including, but not limited to, a step of obtaining the toxin in the supernatant after the above step 4) by centrifugation (12,000×g, 30 minutes).
본 발명의 일 구현 예에서, 상기 해파리 독소는 1-메틸-4-페닐피리디늄 아이오다이드(1-methyl-4-phenylpyridinium iodide)에 의해 촉진된 신경세포의 사멸을 억제하고, 파킨슨병에 의해 감소된 운동능력을 향상시킬 수 있으며, 파킨슨이 유발된 동물모델의 뇌 조직에서 도파민 전구체 관련 효소인 TH(Tyrosine hydroxylase)의 발현을 증가시킬 수 있지만, 이에 한정하는 것은 아니다. In one embodiment of the present invention, the jellyfish toxin can inhibit neuronal cell death promoted by 1-methyl-4-phenylpyridinium iodide, improve motor ability reduced by Parkinson's disease, and increase the expression of TH (Tyrosine hydroxylase), an enzyme related to a dopamine precursor, in brain tissue of an animal model induced with Parkinson's disease, but is not limited thereto.
본 발명에서의 용어 '예방'이란 본 발명에 따른 약학 조성물의 투여에 의해 파킨슨병의 발병을 억제 또는 지연시키는 모든 행위를 의미하고, '치료'란 상기 약학 조성물의 투여에 의해 파킨슨병의 의심 및 발병 개체의 증상이 호전되거나 이롭게 변경되는 모든 행위를 의미한다. The term 'prevention' in the present invention means any act of inhibiting or delaying the onset of Parkinson's disease by administering the pharmaceutical composition according to the present invention, and 'treatment' means any act of improving or beneficially changing the symptoms of a subject suspected of or suffering from Parkinson's disease by administering the pharmaceutical composition.
본 발명의 약학 조성물은 상기 유효성분 이외에 약학적으로 허용 가능한 담체, 부형제 또는 희석제를 더 포함할 수 있다. 상기 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 포함한 다양한 화합물 혹은 혼합물을 들 수 있다. The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-described effective ingredient. The carrier, excipient and diluent that may be included in the pharmaceutical composition include various compounds or mixtures including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.
제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 유효성분에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.When formulated, it is prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.
본 발명에 따른 상기 약학 조성물의 효과적 투여를 위한 투약 경로는 특별히 제한하지 않고, 적절하게 투약 경로를 채택하여 환자에게 사용될 수 있다. 예를 들어, 경구, 직장, 경피, 비경구(피하, 근육, 혈관), 경막, 국부, 흡입 및 기타의 투여방법이 사용될 수 있다. The route of administration for effective administration of the pharmaceutical composition according to the present invention is not particularly limited, and may be used for a patient by adopting an appropriate route of administration. For example, oral, rectal, transdermal, parenteral (subcutaneous, intramuscular, vascular), epidural, topical, inhalation, and other administration methods may be used.
본 발명에 따른 상기 약학 조성물의 투여형태로는 제제학 분야에 알려진 통상의 방법에 따라 다양한 제형을 통해서 투여할 수 있는데, 바람직한 일례로는 캡슐제, 산제, 과립제, 정제, 현탁액, 에멀젼, 시럽 및 에어로졸 중에서 선택된 어느 하나의 제형으로 제조될 수 있다.The pharmaceutical composition according to the present invention may be administered in various dosage forms according to conventional methods known in the pharmaceutical industry. Preferably, the pharmaceutical composition may be prepared in any one dosage form selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols.
본 발명의 약학 조성물은 약학적으로 유효한 양으로 투여될 수 있는데, 본 발명의 용어 '약학적으로 유효한 양'이란 의학적 예방 또는 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 예방 또는 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 질환의 중증도, 약물의 활성, 환자의 연령, 체중, 건강, 성별, 환자의 약물에 대한 민감도, 사용된 본 발명 조성물의 투여 시간, 투여 경로 및 배출 비율, 치료기간, 사용된 본 발명의 조성물과 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 약학 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적으로 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하다.The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount, and the term 'pharmaceutically effective amount' of the present invention means an amount sufficient to prevent or treat a disease at a reasonable benefit/risk ratio applicable to medical prevention or treatment, and the effective dosage level can be determined according to factors including the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the administration time of the composition of the present invention used, the administration route and excretion rate, the treatment period, the drug used in combination or simultaneously with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered singly or in multiple doses. It is important to consider all of the above factors and administer an amount that can obtain the maximum effect with the minimum amount without side effects.
또한, 본 발명은 해파리 독소를 유효성분으로 함유하는 파킨슨병의 예방 또는 개선용 건강기능식품 조성물을 제공한다. In addition, the present invention provides a health functional food composition for preventing or improving Parkinson's disease containing jellyfish toxin as an effective ingredient.
상기 조성물은 분말, 과립, 환, 정제, 캡슐, 캔디, 시럽 및 음료 중에서 선택된 어느 하나의 제형으로 제조될 수 있지만, 이에 한정하는 것은 아니다. The above composition can be prepared in any one dosage form selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage, but is not limited thereto.
본 발명의 건강기능식품 조성물을 식품첨가물로 사용하는 경우, 상기 건강기능식품 조성물을 그대로 첨가하거나 다른 식품 또는 식품성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 양은 그의 사용 목적(예방 또는 개선)에 따라 적절하게 사용될 수 있다. When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to a conventional method. The amount of the effective ingredient may be used appropriately depending on the purpose of use (prevention or improvement).
상기 건강기능식품의 종류에 특별한 제한은 없다. 상기 건강기능식품 조성물을 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There is no special limitation on the type of the above health functional food. Examples of foods to which the above health functional food composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea drinks, alcoholic beverages, and vitamin complexes, and include all health foods in the conventional sense.
또한, 본 발명의 건강기능식품 조성물은 식품, 특히 기능성 식품으로 제조될 수 있다. 본 발명의 기능성 식품은 식품 제조 시에 통상적으로 첨가되는 성분을 포함하며, 예를 들어, 단백질, 탄수화물, 지방, 영양소 및 조미제를 포함한다. 예컨대, 드링크제로 제조되는 경우에는 유효성분 이외에 천연 탄수화물 또는 향미제를 추가 성분으로서 포함할 수 있다. 상기 천연 탄수화물은 모노사카라이드(예컨대, 글루코오스, 프럭토오스 등), 디사카라이드(예컨대, 말토스, 수크로스 등), 올리고당, 폴리사카라이드(예컨대, 덱스트린, 시클로덱스트린 등) 또는 당알코올(예컨대, 자일리톨, 소르비톨, 에리쓰리톨 등)인 것이 바람직하다. 상기 향미제는 천연 향미제(예컨대, 타우마틴, 스테비아 추출물 등)와 합성 향미제(예컨대, 사카린, 아스파르탐 등)를 이용할 수 있다.In addition, the health functional food composition of the present invention can be manufactured as a food, particularly a functional food. The functional food of the present invention includes ingredients that are usually added during food manufacturing, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when manufactured as a drink, it may include natural carbohydrates or flavoring agents as additional ingredients in addition to the effective ingredient. The natural carbohydrate is preferably a monosaccharide (e.g., glucose, fructose, etc.), a disaccharide (e.g., maltose, sucrose, etc.), an oligosaccharide, a polysaccharide (e.g., dextrin, cyclodextrin, etc.), or a sugar alcohol (e.g., xylitol, sorbitol, erythritol, etc.). The flavoring agent can use a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) and a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).
상기 건강기능식품 조성물 이외에 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 더 함유할 수 있다. 이러한 상기 첨가되는 성분의 비율은 크게 중요하진 않지만 본 발명의 건강기능식품 조성물 100 중량부에 대하여, 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above health functional food composition, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. may be further contained. The ratio of these added components is not particularly important, but is generally selected in the range of 0.01 to 0.1 parts by weight with respect to 100 parts by weight of the health functional food composition of the present invention.
또한, 본 발명은 해파리 독소를 유효성분으로 함유하는 파킨슨병의 예방 또는 치료용 수의학적 조성물을 제공한다. In addition, the present invention provides a veterinary composition for preventing or treating Parkinson's disease containing jellyfish toxin as an active ingredient.
본 발명의 수의학적 조성물은 통상의 방법에 따른 적절한 부형제 및 희석제를 더 포함할 수 있다. 본 발명의 수의학적 조성물에 포함될 수 있는 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시 벤조에이트, 탈크, 마그네슘 스테아레이트, 세탄올, 스테아릴알콜, 유동파라핀, 솔비탄모노스테아레이트, 폴리소르베이트 60, 메칠파라벤, 프로필파라벤 및 광물유를 들 수 있다. The veterinary composition of the present invention may further comprise suitable excipients and diluents according to conventional methods. Excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxy benzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil.
본 발명에 따른 수의학적 조성물은 충진제, 항응집제, 윤활제, 습윤제, 향신료, 유화제, 방부제 등을 추가로 포함할 수 있는데, 본 발명에 따른 수의학적 조성물은 동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 잘 알려진 방법을 사용하여 제형화될 수 있고, 제형은 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 용액, 시럽, 에어로졸, 연질 또는 경질 젤라틴 캅셀, 좌제, 멸균 주사용액, 멸균 외용제 등의 형태일 수 있다. The veterinary composition according to the present invention may additionally contain fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, etc., and the veterinary composition according to the present invention may be formulated using methods well known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to an animal, and the formulation may be in the form of a powder, granule, tablet, capsule, suspension, emulsion, solution, syrup, aerosol, soft or hard gelatin capsule, suppository, sterile injectable solution, sterile external preparation, etc.
본 발명에 따른 수의학적 조성물의 유효한 양은 동물의 개체에 따라 적절하게 선택할 수 있다. 질환 내지 상태의 중증도, 개체의 연령, 체중, 건강상태 또는 성별에 따른 본 발명의 유효성분에 대한 민감도, 투여 경로, 투여 기간, 상기 조성물과 배합 또는 동시 사용되는 다른 조성물을 포함한 요소 및 기타 생리 내지 수의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.The effective amount of the veterinary composition according to the present invention can be appropriately selected depending on the individual animal. It can be determined based on factors including the severity of the disease or condition, the sensitivity to the effective ingredient of the present invention according to the age, weight, health status or sex of the individual, the route of administration, the period of administration, other compositions combined or used simultaneously with the composition, and other factors well known in the field of physiology or veterinary medicine.
또한, 본 발명은 해파리 독소를 유효성분으로 함유하는 파킨슨병의 예방 또는 개선용 사료 첨가제를 제공한다. In addition, the present invention provides a feed additive for preventing or improving Parkinson's disease containing jellyfish toxin as an effective ingredient.
상기 유효성분은 파킨슨의 예방 또는 개선을 목적으로 사료 조성물로 첨가할 수 있다. 상기 사료용 조성물은 사료 첨가제를 포함할 수 있다. 본 발명의 사료첨가제는 사료관리법상의 보조사료에 해당한다.The above effective ingredient can be added to a feed composition for the purpose of preventing or improving Parkinson's disease. The feed composition can include a feed additive. The feed additive of the present invention corresponds to a supplementary feed under the Feed Management Act.
본 발명에서 용어 '사료'는 동물이 먹고, 섭취하며, 소화시키기 위한 또는 이에 적당한 임의의 천연 또는 인공 규정식, 한끼식 등 또는 상기 한 끼 식의 성분을 의미할 수 있다. 상기 사료의 종류는 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용되는 사료를 사용할 수 있다. 상기 사료의 비 제한적인 예로는, 곡물류, 근과류, 식품 가공 부산물류, 조류, 섬유질류, 제약 부산물류, 유지류, 전분류, 박류 또는 곡물 부산물류 등과 같은 식물성 사료; 단백질류, 무기물류, 유지류, 광물성류, 유지류, 단세포 단백질류, 동물성 플랑크톤류 또는 음식물 등과 같은 동물성 사료를 들 수 있다. 이들은 단독으로 사용되거나 2종 이상을 혼합하여 사용될 수 있다.In the present invention, the term 'feed' may mean any natural or artificial diet, meal, etc., or ingredients of said meal, which are suitable for animals to eat, ingest, and digest. The type of said feed is not particularly limited, and feed commonly used in the relevant technical field may be used. Non-limiting examples of said feed include plant-based feeds such as grains, roots, food processing by-products, algae, fibers, pharmaceutical by-products, fats, starches, meal, or grain by-products; and animal-based feeds such as proteins, inorganic substances, fats, minerals, fats, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.
이하, 제조예 및 실시예를 이용하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 제조예 및 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들에 의해 제한되지 않는다는 것은 당해 기술분야에서 통상의 지식을 가진 자에게 있어 자명한 것이다. Hereinafter, the present invention will be described in more detail using manufacturing examples and examples. These manufacturing examples and examples are only intended to explain the present invention more specifically, and it is obvious to those skilled in the art that the scope of the present invention is not limited by them.
제조예 1. 해파리 독소 추출Manufacturing Example 1. Extraction of jellyfish toxin
2021년 8월 대한민국 삼천포 연안 해역에서 작은상자 해파리(Carybdea brevipedalia)를 채집한 후, 촉수만 분리한 다음 즉시 얼음에 넣었다. 그 후, 해부된 촉수를 4℃의 깨끗한 바닷물(4% sea salt; 시그마 알드리치 S9883)로 헹구어 잔해물을 제거한 다음 촉수 1 중량부 대비 3 중량부의 깨끗한 바닷물(4% sea salt; 시그마 알드리치 S9883)을 넣고 4℃에서 부드럽게 회전시키며 24시간 동안 자가분해시켰다. 24시간 후, 원심분리(1,000×g, 10분)하여 자포(nematocyst)가 포함된 상등액을 수집한 후, 상등액을 -70℃에서 보관하였고, 다시 남아있는 촉수에 바닷물을 첨가한 후, 24시간 동안 추가로 자가분해를 수행하였다. 상기 과정을 3~4일 동안 반복한 후, 획득된 상등액을 동결건조하여 획득된 자포(nematocyst)를 분말화한 후, -70℃에서 보관하였다.In August 2021, small box jellyfish ( Carybdea brevipedalia ) were collected from the coastal waters of Samcheonpo, South Korea, and only the tentacles were separated and immediately placed on ice. The dissected tentacles were rinsed with clean seawater (4% sea salt; Sigma Aldrich S9883) at 4°C to remove debris. Then, 3 parts by weight of clean seawater (4% sea salt; Sigma Aldrich S9883) was added per 1 part by weight of the tentacles and autolyzed for 24 h with gentle rotation at 4°C. After 24 h, the supernatant containing nematocysts was collected by centrifugation (1,000 × g, 10 min). The supernatant was stored at -70°C, and seawater was added to the remaining tentacles, followed by additional autolyzation for 24 h. After repeating the above process for 3 to 4 days, the obtained supernatant was freeze-dried to obtain nematocysts, which were then powdered and stored at -70°C.
상기 동걸 건조된 자포(nematocyst) 분말 70mg에 차가운 인산완충식염수(PBS, pH7.4, 4℃) 1mL 및 유리구슬(glass beads, 직경 0.5mm)을 혼합한 후, 3000rpm으로 40초 동안 진탕하여 물리적인 충격을 가한 다음 얼음을 이용해 1분간 냉각시키는 과정을 10회 반복하여 독소를 추출하였다. 그 후, 추출된 자포세포 잔해물과 독소를 분리하기 위해 4℃에서 30분 동안 원심분리(12000×g)한 다음 상층액의 독소만 분리하여 해파리 독소(CbV)로 사용하였다. After mixing 70 mg of the dried nematocyst powder with 1 mL of cold phosphate buffered saline (PBS, pH 7.4, 4°C) and glass beads (diameter 0.5 mm), the mixture was shaken at 3000 rpm for 40 seconds to apply physical impact, and then cooled with ice for 1 minute. This process was repeated 10 times to extract the toxin. After that, in order to separate the extracted nematocyst cell debris and the toxin, centrifugation (12000×g) was performed at 4°C for 30 minutes, and only the toxin in the supernatant was separated and used as jellyfish toxin (CbV).
실시예 1. SH-SY5Y에 MPP+를 처리하여 유도된 신경 독성모델에서, 해파리 독소 처리에 따른 세포생존율 변화 확인Example 1. Confirmation of changes in cell viability according to jellyfish toxin treatment in a neurotoxicity model induced by MPP+ treatment in SH-SY5Y
1) SH-SY5Y에서, 해파리 독소(CbV) 또는 MPP+의 세포독성 확인1) Confirmation of cytotoxicity of jellyfish toxin (CbV) or MPP+ in SH-SY5Y
신경모세포종(human neuroblastoma) 유래 세포주인 SH-SY5Y는 시험관 내 파킨슨병 모델을 확립하기 위해 사용하였다. SH-SY5Y 세포는 37℃, 5% CO2 농도의 가습 인큐베이터에서, 10%(v/v) FBS, 100U/mL 페니실린(penicillin) 및 100㎍/mL 스트렙토마이신(streptomycin)이 포함된 DMEM을 사용하여 배양하였다. SH-SY5Y, a human neuroblastoma cell line, was used to establish an in vitro Parkinson's disease model. SH-SY5Y cells were cultured in DMEM containing 10% (v/v) FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C and 5% CO 2 concentration.
세포독성을 확인하기 위해, 1×105 세포수/mL의 밀도로 24웰 플레이트에 세포를 분주한 후, 24시간 동안 배양한 다음, 다양한 농도의 CbV 및 MPP+를 처리하여 24시간 동안 재배양하였다. 24시간 후, MTT 시약을 각 웰에 50㎕씩 처리하였고, 3시간 동안 반응시킨 후, 배지를 제거하고, 500㎕의 DMSO를 첨가하여 보라색 포마진을 용해시켰고, 마이크로플레이트 리더를 이용하여 540nm에서 흡광도를 측정하였다. To examine cytotoxicity, cells were seeded in a 24-well plate at a density of 1 × 10 5 cells/mL, cultured for 24 h, and then treated with various concentrations of CbV and MPP+ and re-cultured for 24 h. After 24 h, 50 μl of MTT reagent was treated to each well, and after reaction for 3 h, the medium was removed, 500 μl of DMSO was added to dissolve the purple formazin, and the absorbance was measured at 540 nm using a microplate reader.
세포독성은 세포생존율로 확인하였고, 세포생존율은 하기 식 1과 같이, 정상군의 값을 control로 하고 이때의 O.D.(Optical density)값을 세포의 생존도가 100% 라고 정의하고, 나머지 군의 O.D. 값을 상대치로 환산하여 계산하였다. 결과는 3회 반복 실험한 평균값으로 나타냈다. Cytotoxicity was confirmed by cell viability, and cell viability was calculated as shown in Equation 1 below, with the value of the normal group as the control, the O.D. (Optical density) value at this time being defined as 100% cell viability, and the O.D. values of the remaining groups were converted to relative values and calculated. The results were expressed as the average value of three repeated experiments.
[식 1][Formula 1]
세포생존율=(실험군 O.D. 값/ 정상군 O.D. 값)×100 Cell viability = (O.D. value of experimental group / O.D. value of normal group) × 100
그 결과, 도 1에 개시된 바와 같이 해파리 독소(CbV)는 3ng/mL까지 세포생존율에 전혀 영향을 미치지 않아, 이후 실험에서는 해파리 독소를 3ng/mL 농도까지 사용하였다. As a result, as disclosed in Fig. 1, jellyfish toxin (CbV) had no effect at all on cell viability up to 3 ng/mL, so jellyfish toxin was used at a concentration of up to 3 ng/mL in subsequent experiments.
MPP+는 2mM 처리시 세포생존율이 50%로 나타나, 이후 실험에서는 MPP+의 처리 농도를 2mM로 사용하였다. MPP+ showed a cell viability of 50% when treated with 2 mM, so in subsequent experiments, 2 mM MPP+ was used.
2) SH-SY5Y에 MPP+를 처리하여 유도된 신경 독성모델에서, 해파리 독소(CbV) 처리에 따른 세포보호 효과 확인2) In a neurotoxicity model induced by treating SH-SY5Y with MPP+, cytoprotective effects were confirmed by treatment with jellyfish toxin (CbV).
세포보호 효과는 세포생존율로 확인하였다. The cytoprotective effect was confirmed by cell viability.
상기와 동일한 조건으로 1×105 세포수/mL의 밀도로 24웰 플레이트에 분주한 후, 24시간 동안 배양한 후, 2mM MPP+를 3시간 동안 전처리한 뒤 CbV를 0.3, 1 및 3ng/mL 농도로 처리한 후 24시간 동안 배양하였다. 그 후, 광학 현미경을 통해 세포를 관찰한 다음 상기와 동일한 방법으로 세포생존율을 확인하였다. After seeding into a 24-well plate at a density of 1×10 5 cells/mL under the same conditions as above, the cells were cultured for 24 hours, pretreated with 2 mM MPP+ for 3 hours, and then treated with CbV at concentrations of 0.3, 1, and 3 ng/mL, followed by culture for 24 hours. Thereafter, the cells were observed through an optical microscope and the cell viability was confirmed using the same method as above.
그 결과, 도 2에 개시된 바와 같이 광학 현미경 하에서 SH-SY5Y 세포에 MPP+가 처리된 그룹에서 세포 사멸과 관련된 형태학적 변화인 세포 수축 및 퇴화가 관찰되었고, CbV를 함께 처리한 그룹에서는 MPP+에 의해 유도된 형태학적 변화가 개선되는 것을 확인하여, CbV가 MPP+에 의해 유도된 세포독성에 대한 세포보호 효과를 가져 세포생존율이 향상되는 것을 확인하였다. As a result, as disclosed in Fig. 2, cell shrinkage and degeneration, which are morphological changes associated with cell death, were observed in the group treated with MPP+ in SH-SY5Y cells under an optical microscope, and in the group treated with CbV together, it was confirmed that the morphological changes induced by MPP+ were improved, confirming that CbV has a cytoprotective effect against cytotoxicity induced by MPP+, thereby improving cell viability.
3) DAPI 염색3) DAPI staining
세포독성과 세포자살(apoptosis)이 관련이 있는지를 확인하기 위해, DAPI 염색을 통해 핵 염색을 실시하였다.To determine whether cytotoxicity and apoptosis were related, nuclear staining was performed using DAPI staining.
상기와 동일한 조건으로 1×105 세포수/mL의 밀도로 24웰 플레이트에 분주한 후, 24시간 동안 배양한 다음 2mM MPP+를 3시간 동안 전처리한 뒤 CbV를 0.3, 1 및 3ng/mL 농도로 처리한 후 24시간 동안 배양하였다. 그 후, 세포를 DAPI(4',6-diamidino-2-phenylindole)로 염색한 후, 세포 형태 및 사멸 정도를 확인하였다. After seeding into a 24-well plate at a density of 1×10 5 cells/mL under the same conditions as above, the cells were cultured for 24 h, pretreated with 2 mM MPP+ for 3 h, and then treated with CbV at concentrations of 0.3, 1, and 3 ng/mL, and cultured for 24 h. After that, the cells were stained with DAPI (4',6-diamidino-2-phenylindole), and the cell morphology and degree of death were confirmed.
구체적으로, 세포를 PBS로 세척한 후, 실온에서 10분 동안 4%(v/v) 포름알데히드로 고정한 후, 5분 동안 PBS에서 Triton X-100 0.2%(v/v)와 함께 인큐베이션한 다음, 세포를 실온에서 15분 동안 DAPI(1㎍/mL)와 함께 인큐베이션한 후, 커버-슬립핑하고 현미경(Termo)으로 관찰하였다. 세포자살(apoptosis) 세포의 형태학적 변화는 DAPI 염색에 의해 가시화되었고, 대표적인 이미지는 200x 대물렌즈를 사용하여 현미경(Termo)으로 포착되었다. DAPI 양성 세포는 현미경 하에서 400x 배율로 무작위로 선택된 10개의 필드에서 정량화한 후 계산하였다. Specifically, cells were washed with PBS, fixed with 4% (v/v) formaldehyde for 10 min at room temperature, incubated with 0.2% (v/v) Triton X-100 in PBS for 5 min, and then incubated with DAPI (1 μg/mL) for 15 min at room temperature, cover-slipped, and observed under a microscope (Termo). Morphological changes of apoptotic cells were visualized by DAPI staining, and representative images were captured under a microscope (Termo) using a 200x objective. DAPI-positive cells were quantified and then counted in 10 randomly selected fields at 400x magnification under a microscope.
그 결과, 도 3에 개시된 바와 같이 SH-SY5Y 세포에 CbV 만을 처리한 그룹에서는 핵 모양의 변화 및 세포자살이 일어나지 않았으나, MPP+만 처리된 그룹에서는 핵이 깨어지고 응축되었으며, 핵 크기 역시 매우 감소하여 세포자살이 일어난 세포수가 크게 증가됨을 확인하였다. 반면, MPP+ 및 CbV를 처리한 그룹에서는 농도 의존적으로 세포자살이 일어난 세포수가 크게 줄어들었다.As a result, as disclosed in Fig. 3, in the group treated with only CbV in SH-SY5Y cells, no change in nuclear shape or apoptosis occurred, but in the group treated with only MPP+, the nuclei were broken and condensed, and the nuclear size was also greatly reduced, confirming that the number of cells that underwent apoptosis greatly increased. On the other hand, in the group treated with MPP+ and CbV, the number of cells that underwent apoptosis greatly decreased in a concentration-dependent manner.
실시예 2. SH-SY5Y에 MPP+를 처리하여 유도된 신경 독성모델에서, 해파리 독소(CbV) 처리에 따른 TH(Tyrosine hydroxylase) 발현 변화 확인Example 2. Confirmation of changes in TH (Tyrosine hydroxylase) expression following jellyfish toxin (CbV) treatment in a neurotoxicity model induced by MPP+ treatment in SH-SY5Y
TH(Tyrosine hydroxylase)는 도파민 생합성의 속도 제한 효소이며, TH 발현의 감소는 도파민 합성 감소를 초래하고 파킨슨병으로 이어진다. 따라서 해파리 독소 처리에 따른 도파민 전구체 관련 효소인 TH의 단백질 발현 변화를 웨스턴 블롯으로 확인하였다. TH (Tyrosine hydroxylase) is the rate-limiting enzyme for dopamine biosynthesis, and a decrease in TH expression leads to a decrease in dopamine synthesis and Parkinson's disease. Therefore, the change in protein expression of TH, an enzyme related to the dopamine precursor, following jellyfish venom treatment was confirmed by Western blot.
구체적으로, 프로테아제 억제제 칵테일을 포함하는 RIPA(radioimmunoprecipitation assay) 완충액(TransLab, Daejeon, Korea)을 사용하여 세포로부터 단백질을 추출한 후, 브래드 포드 분석을 통해 단백질 농도를 측정하였다. 동량의 단백질 샘플을 SDS-PAGE 샘플 버퍼(62.5mM Tris-HCl pH 6.8, 10% 글리세롤, 2% SDS, 0.01% 브로모페놀 블루)와 혼합하고 10분 동안 끓인 후, 각 샘플을 12% SDS-PAGE(sodium dodecylsulfate-polyacrylamide gradient gel) 영동법으로 분리하였고, PVDF(polyvinylidene fluoride) 멤브레인으로 옮겼다. 멤브레인은 5%(w/v) BSA에서 블로킹(blocking)한 다음, 4℃에서 하룻밤 동안 1차 항체로 반응시킨 후, TBS-T에서 10분씩 3회 세척하였다. 그 후, 2차 항체와 함께 1시간 동안 반응시킨 다음 세척한 뒤 ECL(enhanced chemiluminiscence system, Amersham Biosciences, UK)로 시각화하고, Chemi doc XRS(Bio-Rad, CA USA)를 사용하여 분석하였다. 단백질 밴드 분석은 Hewlett-Packard 스캐너와 NIH Image 소프트웨어(Image J, National Institutes of Health, Bethesda, MD, USA)를 사용하여 수행되었다.Specifically, proteins were extracted from cells using radioimmunoprecipitation assay (RIPA) buffer (TransLab, Daejeon, Korea) containing a protease inhibitor cocktail, and then protein concentrations were measured by Bradford assay. Equal amounts of protein samples were mixed with SDS-PAGE sample buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 0.01% bromophenol blue), boiled for 10 min, and each sample was separated by 12% sodium dodecylsulfate-polyacrylamide gradient gel (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 5% (w/v) BSA, reacted with primary antibodies overnight at 4°C, and then washed three times for 10 min each in TBS-T. Afterwards, the membranes were incubated with secondary antibodies for 1 hour, washed, visualized by ECL (enhanced chemiluminiscence system, Amersham Biosciences, UK), and analyzed using Chemi doc XRS (Bio-Rad, CA USA). Protein band analysis was performed using a Hewlett-Packard scanner and NIH Image software (Image J, National Institutes of Health, Bethesda, MD, USA).
그 결과, 도 4에 개시된 바와 같이 아무것도 처리하지 않은 대조군 세포에 비해 MPP+ 단독 처리군은 TH 발현이 유의하게 감소한 반면, MPP+ 및 CbV를 처리한 군은 MPP+ 단독 처리군과 비교하여 TH 발현을 증가시켰다.As a result, as disclosed in Fig. 4, the MPP+ only treatment group showed a significant decrease in TH expression compared to the untreated control cells, whereas the MPP+ and CbV treated group showed an increase in TH expression compared to the MPP+ only treatment group.
실시예 3. 파킨슨병 동물모델에서, 해파리 독소(CbV) 투여에 의한 운동능력 향상 효과 확인-행동분석Example 3. Confirmation of the effect of improving motor ability by administering jellyfish venom (CbV) in an animal model of Parkinson's disease - Behavioral analysis
1) 실험동물 사육1) Breeding of laboratory animals
실험동물은 C57BL/6 마우스(8주령, 21~25g)을 샘타코(Osan, Korea)에서 공급받아 일주일 동안 실험동물실 환경에 적응 및 순화를 시켰다. 동물 사육장의 온도는 22±2℃, 습도는 55±10%를 유지하였으며, 조명은 12시간을 주기로 조절하였다. 물과 고형사료는 자유롭게 섭취하도록 하였다.Experimental animals were C57BL/6 mice (8 weeks old, 21–25 g) supplied by Samtaco (Osan, Korea) and were acclimated to the experimental animal room environment for one week. The temperature and humidity of the animal enclosure were maintained at 22±2℃ and 55±10%, respectively, and lighting was controlled in a 12-hour cycle. Water and solid food were freely available.
2) 파킨슨병 동물모델 확립 및 해파리 독소(CbV) 처리2) Establishment of Parkinson's disease animal model and treatment with jellyfish venom (CbV)
MPTP는 카테콜아민(catecholamine)성 신경만을 선택적으로 파괴하는 신경독성 물질로, 파킨슨병 연구용 동물모델에 널리 사용되는 물질이다. MPTP는 체내에 흡수되어 뇌혈관 장벽(brain-blood barrier)을 통과한 후, 성상 세포(astrocyte)에서 MAO-B(monoamineoxidase B)에 의해 MPP+(1-methyl-4-phenylpyridi-nium)로 대사되어 기저핵의 흑질에 있는 도파민성 신경세포에만 선택적으로 작용한다.MPTP is a neurotoxic substance that selectively destroys only catecholaminergic neurons, and is widely used in animal models for Parkinson's disease research. MPTP is absorbed into the body, passes through the brain-blood barrier, and is metabolized to MPP+ (1-methyl-4-phenylpyridinium) by MAO-B (monoamineoxidase B) in astrocytes, which selectively acts only on dopaminergic neurons in the substantia nigra of the basal ganglia.
C57BL/6 마우스를 난괴법으로 하기 표 1과 같이 5개 그룹(그룹당 6마리)으로 그룹화하였다.C57BL/6 mice were divided into five groups (six mice per group) using the egg block method as shown in Table 1.
정상 대조군은 마우스에 식염수를 14일 동안 매일 복강내(i.p) 주사하였다. MPTP 그룹은 마우스에 연속 5일 동안 매일 30mg/kg의 최종 농도로 MPTP(PBS에 용해됨)를 i.p 주사한 다음, 나머지 9일 동안은 식염수로 처리하였다. MPTP+CbV 또는 L-DOPA 그룹은 마우스에 4일간 CbV 또는 L-DOPA를 전처리 주사하였고, 5일 동안은 주사 1시간 후 MPTP를 투여하였으며, 다음 5일 동안은 CbV 또는 L-DOPA를 각각 i.p 주사하였다. The normal control group was intraperitoneally (i.p.) injected with saline daily for 14 days. The MPTP group was i.p. injected with MPTP (dissolved in PBS) at a final concentration of 30 mg/kg daily for 5 consecutive days, and then treated with saline for the remaining 9 days. The MPTP+CbV or L-DOPA group was pretreated with CbV or L-DOPA for 4 days, then MPTP was administered 1 hour after the injection for 5 days, and then i.p. injected with CbV or L-DOPA, respectively, for the next 5 days.
실험 종료 시점에 행동 테스트를 실시했고, 행동 변화 분석 후, 동물을 마취시켜 희생시키고, 즉시 해부하였다. 실험 일정은 도 5에 도식화한 바와 같다. At the end of the experiment, a behavioral test was conducted, and after analyzing the behavioral changes, the animals were anesthetized, sacrificed, and immediately dissected. The experimental schedule is as diagrammed in Fig. 5.
3) 막대 실험(Pole test)3) Pole test
마우스를 표면이 거친 수직 막대(직경 1cm, 높이 50cm) 위에 놓고 막대 바닥에 도달할 때까지의 시간(초)을 기록하였다. 실제 테스트 전 마우스를 훈련했으며, 세 번 시도의 평균 시간을 기록하였다. Mice were placed on a rough vertical bar (1 cm in diameter, 50 cm high), and the time (in seconds) until they reached the bottom of the bar was recorded. Mice were trained before the actual test, and the average time of three trials was recorded.
그 결과, 도 6에 개시된 바와 같이 정상 대조군 대비 파킨슨병 동물모델에서 증가된 막대 바닥에 도달할 때까지의 시간이 CbV 또는 L-Dopa를 투여한 경우 감소하여 운동능력이 향상된 것을 확인할 수 있었다. As a result, as disclosed in Fig. 6, it was confirmed that the time until reaching the bottom of the rod, which was increased in the Parkinson's disease animal model compared to the normal control group, was reduced when CbV or L-Dopa was administered, indicating improved motor ability.
4) 악력 시험(Grip strength)4) Grip strength test
악력 시험은 악력 측정기(JD-A-22, Jeungdo, Instruments, Inc., Korea)를 사용하여 수행하였다. 마우스 꼬리를 잡고 앞발로 그리드를 잡도록 한 후, 뒷다리가 그리드와 접촉하지 않도록 마우스를 꼬리로 들어올린 후, 더이상 그리드를 잡을 수 없을 때까지 마우스를 꼬리로 부드럽게 뒤로 당겼다. 그립 강도는 그램(g) 단위로 점수화하였고, 3회 평균을 계산하였다.Grip strength tests were performed using a grip strength tester (JD-A-22, Jeungdo, Instruments, Inc., Korea). The mouse was held by the tail and the grid was grasped with the forelimbs. The mouse was lifted by the tail so that the hind limbs did not contact the grid, and the mouse was gently pulled backward by the tail until it could no longer grasp the grid. Grip strength was scored in grams (g), and the average of three attempts was calculated.
그 결과, 도 7에 개시된 바와 같이 정상 대조군 대비 파킨슨병 동물모델에서 감소된 악력이 CbV 또는 L-Dopa를 투여한 경우 다시 증가한 것을 확인하였다. As a result, as disclosed in Fig. 7, it was confirmed that the reduced grip strength in the Parkinson's disease animal model compared to the normal control group increased again when CbV or L-Dopa was administered.
5) 좁은 빔 실험(time on narrow beam test)5) Narrow beam test (time on narrow beam test)
좁은 빔 실험은 모터 조정을 측정하기 위해 수행되었다. 먼저, 마우스를 바닥 위 100cm 높이에 위치한 고정식 목재의 좁은 평면 빔(1cm)에서 움직이도록 훈련한 다음 마우스가 한쪽 끝에서 다른 쪽 끝으로 걷는 시간을 기록하였다. 결과는 각 그룹의 동물에 대해 세 번 반복하여 마우스에 걸린 평균 총 시간(s)으로 표시하였다. The narrow beam experiment was performed to measure motor coordination. First, mice were trained to move on a narrow flat beam (1 cm) of fixed wood placed 100 cm above the floor, and the time taken for the mice to walk from one end to the other was recorded. Results were repeated three times for each group of animals and expressed as the average total time (s) taken by the mice.
그 결과, 도 8에 개시된 바와 같이 정상 대조군 대비 파킨슨병 동물모델에서 마우스가 막대를 내려오기 위해 아래로 향하는 시간이 증가된 반면, CbV 또는 L-Dopa를 투여한 경우 마우스가 막대를 내려오기 위해 아래로 향하는 시간이 다시 감소한 것을 확인하였다. As a result, as disclosed in Fig. 8, it was confirmed that the time taken by the mice to head down the rod increased compared to the normal control group in the Parkinson's disease animal model, whereas when CbV or L-Dopa was administered, the time taken by the mice to head down the rod decreased again.
실시예 4. 파킨슨병 동물모델에서, 해파리 독소(CbV) 투여에 의한 도파민 전구체 관련 효소 증가 효과 확인Example 4. Confirmation of the effect of increasing dopamine precursor-related enzymes by administering jellyfish venom (CbV) in an animal model of Parkinson's disease
1) TH의 면역화학염색1) Immunochemical staining of TH
상기 실시예 3의 행동실험 종료 후, 마우스 뇌 조직을 적출한 후, 즉시 10% 인산염 완충 포르말린으로 24시간 동안 고정하였고, 파라핀 섹션을 처리하였다. 24시간 후, 30% 수크로스(sucrose)에서 24시간을 넣어둔 다음 조직절편기(cryomicrotome)를 이용하여 5μm의 두께로 절단하여 흑질(substantia nigra, SN) 부분이 포함된 부위를 획득하였다. 파라핀 절편을 3% 과산화수소 용액에 넣고 실온의 암실에서 20분 동안 배양한 다음 탈색 진탕기에서 5분 동안 총 3회 세척하였다. 세척 후, 조직을 실온에서 30분 동안 3% BSA와 함께 인큐베이션한 다음 용액을 제거하고 조직 절편을 1차 TH 항체와 함께 4℃에서 16시간 동안 배양하였다. 배양 후, 절편을 PBS에서 3회 세척한 다음, Vectastain Elite ABC kit(Vector Laboratories, Burlingame, CA, USA)를 이용하여 2차 항체와 ABC 시약을 처리한 후 DAB 기질(substrate)을 넣어 발색을 유도한 후, 염색된 도파민 신경세포를 관찰하였다. 섹션에서 양성 항체 발현은 짙은 갈색이었고 이미지는 현미경으로 확인하였다. After the behavioral experiment of Example 3 above was completed, the mouse brain tissue was extracted, immediately fixed in 10% phosphate-buffered formalin for 24 hours, and processed for paraffin sections. After 24 hours, the tissue was placed in 30% sucrose for 24 hours, then sectioned at a thickness of 5 μm using a tissue microtome (cryomicrotome) to obtain the area including the substantia nigra (SN). The paraffin sections were placed in a 3% hydrogen peroxide solution, incubated for 20 minutes in a dark room at room temperature, and then washed three times for 5 minutes each in a decolorizing shaker. After washing, the tissues were incubated with 3% BSA at room temperature for 30 minutes, the solution was removed, and the tissue sections were incubated with primary TH antibodies at 4°C for 16 hours. After incubation, the sections were washed three times in PBS, treated with secondary antibodies and ABC reagent using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA), and then stained with DAB substrate to induce color development, and stained dopamine neurons were observed. Positive antibody expression in the sections was dark brown, and the images were confirmed under a microscope.
SN에서 TH-면역 반응성 뉴런의 수를 확인하였고, Image J(National Institutes of Health, Bethesda, MD, USA)를 사용하여 도파민성 뉴런 섬유의 강도를 정량화하였다.The number of TH-immunoreactive neurons in the SN was determined, and the intensity of dopaminergic neuron fibers was quantified using Image J (National Institutes of Health, Bethesda, MD, USA).
그 결과, 도 9에 개시된 바와 같이 CbV를 투여한 실험군은 MPTP에 의해 감소된 흑질에서의 TH 발현을 파킨슨 질환 치료제인 L-dopa를 투여한 실험군과 유사한 수준으로 유의하게 증가시켰다.As a result, as disclosed in Fig. 9, the experimental group administered CbV significantly increased TH expression in the substantia nigra, which was reduced by MPTP, to a level similar to that of the experimental group administered L-dopa, a treatment for Parkinson's disease.
2) 웨스턴 블롯 분석2) Western blot analysis
파킨슨병 동물모델의 뇌 선조체에서 CbV 투여에 의한 도파민 전구체 관련 효소인 TH의 발현변화를 웨스턴 블롯 분석으로 확인하였다. 웨스턴 블롯 분석은 실시예 2와 동일한 방법으로 진행하였다.The change in the expression of TH, an enzyme related to the dopamine precursor, in the brain striatum of an animal model of Parkinson's disease was confirmed by Western blot analysis due to CbV administration. Western blot analysis was performed in the same manner as in Example 2.
그 결과, 도 10에 개시된 바와 같이 CbV를 투여한 실험군은 MPTP에 의해 감소된 TH의 발현을 농도의존적으로 증가시켰다. As a result, as disclosed in Fig. 10, the experimental group administered CbV increased the expression of TH, which was reduced by MPTP, in a concentration-dependent manner.
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