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Kafe 바로가기주관연구기관 | 국립농업과학원 National Institute of Agricultural Sciences |
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보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 | 한국어 |
발행년월 | 2016-02 |
과제시작연도 | 2015 |
주관부처 | 농촌진흥청 Rural Development Administration(RDA) |
과제관리전문기관 | 국립농업과학원 National Institute of Agricultural Sciences |
등록번호 | TRKO201600003227 |
과제고유번호 | 1395039908 |
사업명 | 농업기초기반연구 |
DB 구축일자 | 2016-06-25 |
DOI | https://doi.org/10.23000/TRKO201600003227 |
Ⅳ. 연구개발결과
제1세부 : 곤충 프로테오글라이칸 순환기 개선물질 약리독성연구
구조동정: 곤충프로테오글라이칸(글라이코자미노글라이칸을 포함) 7종의 구조동정을 위하여 일차원적, 2차원적 프로톤 NMR 분석을 실시하였으며 글라이칸 가수분해효소 중 헤파리나제 I, II, III로 효소분해하여 저분자화하여 LCMS을 하여 구조분석을 실시하여 표준품대비 LCMS data base를 구축. ① 강랑(쇠똥구리), ② 귀뚜라미, ③ 매미눈꽃동충하초, ④ 맹충(쇠등에), 및 ⑤ 광대노린재, ⑥호박벌여왕벌, ⑦호박벌봉군껍질, ⑧서양뒤영벌
Ⅳ. 연구개발결과
제1세부 : 곤충 프로테오글라이칸 순환기 개선물질 약리독성연구
구조동정: 곤충프로테오글라이칸(글라이코자미노글라이칸을 포함) 7종의 구조동정을 위하여 일차원적, 2차원적 프로톤 NMR 분석을 실시하였으며 글라이칸 가수분해효소 중 헤파리나제 I, II, III로 효소분해하여 저분자화하여 LCMS을 하여 구조분석을 실시하여 표준품대비 LCMS data base를 구축. ① 강랑(쇠똥구리), ② 귀뚜라미, ③ 매미눈꽃동충하초, ④ 맹충(쇠등에), 및 ⑤ 광대노린재, ⑥호박벌여왕벌, ⑦호박벌봉군껍질, ⑧서양뒤영벌여왕벌, ⑨일벌에서 MALDI-TOF MS/MS n- 글라이칸 당서열 동정, 귀뚜라미 글라이코자미노글라이칸 분해효소(헤파리나제I, II, III)와 벤조나제 처리 저분자 가수분해물동정: 헤파린 이당류 IV-H, I-S, I-H 및 분해물 MS/MS확인 동정
In vitro assay: 곤충 N-glycan 항응고활성과 혈관부착물질 라미닌과 파이브로넥틴 변동ghkrdls
In Vivo assay: 노화쥐와 고지방식이쥐에 대한 귀뚜라미알콜추출물, 귀뚜라미 글라이코자미노 글라이칸에 대한 순환기개선효과에 대한 순환기개선효과 동물전임상실험 수행 결과 고지혈증개선 지방간억제, 항노화 등 시판제품 대비 효과적인 순환기개선 결과를 얻었다.
즉, GAG 분해 이당류구조 , N-글라이칸 단당 서열 구명, 13주 귀뚜라미 분말에서 체지방축적 억제 효과를 얻었다. 항노화 효과(anti-aginging effect)를 양성대조인 블루베리 알콜 추출물과 비교하면서 동량의 귀뚜라미 알콜추출물(100 mg/kg) 투여하여 8-OHdG의 DNA손상체 혈중 감소와 CK 억제를 확인하였다. 귀뚜라미 알콜추출물(100 또는 200 mg/kg), IS ex(100 mg/kg), 양성대조군: Statin(2 mg/kg) 양성대조 스타틴과 비교 한 달/두 달 경구투여하여 고지방식이를 실시한 60% HFD Wistar 랫트에 고지혈증, 지방간 개선효과를 확인[한달 투여로 카타라제(지질손상지표), IL-10활성, 2 달 투여로카타라제, 카르보닐함량(단백질 손상지표)감소] GbGAG고지혈, 지방간 억제 확인시험으로 1주 60% HFD를 전처치한 Wistar rat에 다시 고비방사료를 식이하면서 GbGAG (5 or 10 mg/kg)를 한달 복강 투여하여 고지혈증(혈당감소)개선과 순환기개선작용(염증에 관여 CRP, AST, ALT 감소)를 확인하였다.
CON, GbGAG, ISGAG, IQGAG, STA의 6주간 60% HFD 전처치한 Wistar rat에 한달 경구투여. 체지방, 혈관내피(라미닌, 파이브로넥틴) 부착억제를 확인하였다.
제2세부 : 곤충 귀뚜라미 순환기개선효과구명 및 식품화연구
귀뚜라미의 순환기 개선작용을 귀뚜라미분획(헥산, 클로르포름, 에칠아세테이트, 부탄올 분획)에 대하여 발부봉, 염증 유발한 SD 대하여 발부종감소와 혈청중의 분비되는 사이토카인의 농도를 비교한 결과 귀뚜라미 글라이코자미노글라이칸 10 mg/kg과 일부분획에서 유리지방산의 감소, 부탄올분획의 RA factor의 감소, triglyceride 감소가 관찰되었다. 즉, 보체유도 시간에서 5일에 걸쳐 발부종의 감소비율은 분획별로: ethanol 분획 (32.35%), butanol 분획 (0.48%), ethyl acetate 분획 (18.23%), chloroform 분획 (15.44%), hexane 분획 (16.08%)과 Gb-GAG (32.55%)이었다. 귀뚜라미 알콜추출물의 4주간 반복경구투여독성시험에서 4주간 귀뚜라미 알콜추출물을 쥐에 투여시 섭취량 체중변화에 별다른 임상증세를 관찰할 수 없었으며, 무독성량(NOAEL) : 1,000 ㎎/㎏/day, 일일섭취허용량(ADI) : 600 ㎎/man/day으로 산출되었다. 노화쥐에 귀뚜라미 알콜추출물을 한달 투여하여 1053 유전자 발현이 증가하였고 그 중 Fas,Amigo3, Rtn4 dl이 발현이 증가하였고, Ugt2b (UDP glycosyltransferase 2 family), Egr1, Gpm6a등 73개 유전자 발현이 감소하였다.
The insect-derived proteoglycans relating to the circulatory system and their pharmacological study
Structural analysis
The structures of proteoglycans, including glycosaminoglycans (GAGs), derived from 7 species of insects (9 different insects), were determined using 2D proton NMR, LC-MS (MAL
The insect-derived proteoglycans relating to the circulatory system and their pharmacological study
Structural analysis
The structures of proteoglycans, including glycosaminoglycans (GAGs), derived from 7 species of insects (9 different insects), were determined using 2D proton NMR, LC-MS (MALDI-TOF), and LC-MS/MS on small molecular weight GAGs using glycan lyase digestion (heparinase I, II, III). Heparin disaccharide, IV-H, I-S, and I-H digestion products, or N-glycan sugar sequences were detected from ① Dung beetle (Catharsius molossus), ② Cricket (Gryllus bimaculatus), ③ Isaria sinclairii, ④ Tabanus (Tabanus bivittatus), ⑤ Poecilocoris lewisi (Kwangdaenolinjae), ⑥ Queen of Bumbus ignitus, ⑦ Colony shell of B. ignitus (BIC), ⑧ Queen of B. terrestris, and ⑨ B. terrestris worker by mass spectrometry.
In vivo assay
To ascertain the anti-aging activity, 14-month-old WKY rats were treated with the ethanol extract (100 mg/kg) of Gryllus bimaculatus (Gb, a type of cricket), and the reduction in the DNA oxidative damage marker, 8-OHdG, and creatinine kinase enzyme activity, in serum were assessed and compared with the ethanol extract of blueberry (100 mg/kg). Furthermore, a 60% high-fat diet (HFD) was given to induce hyperlipidemia in Wistar rats, as an efficacy test for the antilipidemic effect of Gb EtOH extract or Gb glycosaminoglycans compared with Pravastatin or chitosan, as commercial drugs. There were relatively significant antilipidemic activities seen in HFD-fed rats as well as the anti-aging activity in WKY aged rats. Gb EtOH extract showed a reduction in the levels of a protein oxidative damage marker and carbonyl content, and an increase in IL-10, following 1 month of treatment, whereas there was an induction of catalase activity and a reduction in carbonyl content following 2 months of treatment, compared with Isaria sinclairii extract (100 mg/kg) and Pravastatin (2 mg/kg) in HFD-fed rats (1 month HFD pretreatment, gene expression profiling). Moreover, Gb GAG (5 or 10 mg/kg) was shown to ameliorate hyperglycemia and blood circulation, with an anti-inflammatory effect, as seen by a reduction in CRP, AST, and ALT activities in HFD-fed (1 week HFD pretreatment) rat serum compared with statin (2 mg/kg) or chitosan (10 mg/kg) over one month with gene expression profiling. Several insect GAGs: GbGAG, ISGAG, and IQGAG were also tested in HFD-fed (6 weeks HFD pretreatment) rats with chronic hyperglycemia. Adipose tissue weight was reduced, and there was an inhibition of adhesion via laminin or fibronectin (ECM binding molecules) seen with each GAG.
The effect of cricket extract on the circulatory system and its food study
Anti-inflammatory effect of Gb extract
The water extract, as well as the ethanol extract, showed an anti-edema effect in a CFA-treated rat model of arthritis. To identify the active compounds in the extracts of Gb, 5 groups were orally treated with each sample (100 mg/kg) for 5 days. The anti-edema effect of the individual extracts on the rat paws was as follows: Gb glycosaminoglycans > ethanol extract > ethyl acetate or chloroform or hexane fraction > butanol fraction. The glycosaminoglycans of G. bimaculatus (Gb GAG) were further purified from crude Gb GAG through decolorization using DEAE GPC, which also displayed anti-edema effects. The mean percentage decrease in paw edema size (mm) from 1 h to day 5 for each group was as follows: EtOH ex. (32.35%), BuOH fr. (0.48%), EtOAC fr. (18.23%), CHCl3fr.(15.44%), Hexane fr.(16.08%), and GbGAG (32.55%).
4-week oral dose toxicity study of Gb ethanol extract
For certification of the safety margin of Gb extract, at doses of 0, 0.25, 5, and 1 g/kg for 4 consecutive weeks, there were no observed clinical signs or deaths related to treatment in any of the groups tested.
The approximate lethal oral dose of Gb ex. was considered to be higher than 1 g/kg in rats. Microscopic examination did not identify any treatment-related histopathological changes in the organs of the Gb ex-treated rats in the high dose group. From these results, it can be concluded that the no-observed effect level (NOAEL) of G. bimaculatus is less than 1 g/kg in rats, and the ADI is 600 mg/man/day.
Gene expression profiling of aged Wistar rats treated with Gb extract
G. bimaculatus extract may have value in lessening the effects of aging, resulting in a differential gene expression pattern indicative of a marked stress response and lower expression of metabolic and biosynthetic genes. Compared with untreated control rats, rats treated with the extract displayed upregulation of 1053 genes including Amigo3, Hmgcr (a reductase), and Rtn (an antioxidant), and downregulation of 73 genes including UDP glycosyltransferase and glycoprotein m6a.
Safety of cricket extrcat as a food
For certification of safety margin of Gb extract, at doses of 0, 0.25, 5, and 1 g/kg for 4 consecutive weeks, there were no observed clinical signs or deaths related to treatment in all the groups tested. The approximate lethal oral dose of G b ex. was considered to be higher than 1 g/kg in rats. Microscopic examination did not show to identify any treatment-related histopathologic changes in the organs of the Gb ex-treated rats in the high dose group. From these results, one can conclude that the no-observed effect level (NOAEL) of G. bimaculatus is less than 1 g/kg in rats and ADI is 600 mg/man/day.
Gene expression profiling of cricket- treated rat liver
G. bimaculatus extract may have value in lessening the effects of aging, resulting in a differential gene expression pattern indicative of a marked stress response and lower expression of metabolic and biosynthetic genes. Compared with untreated control rats, rats treated with the extract displayed the upregulation of 1053 genes including, Amigo3, Hmgcr; a reductase, and Rtn antioxidant, and the downregulation of 73 genes including UDP glycosyltransferase, and Glycoprotein m6a.
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