홍화씨 및 약용식물 추출물과 복합제제의 체내 지질상태 개선 효과 = Improvement of body lipid status by extracts from safflower seed and medicinal plants and their formulated product

다국어 초록 (Multilingual Abstract)

This study was carried out to evaluate the lipid modulating effects of extracts from safflower seed and a few traditional medicinal plants and to examine the possibility of utilizing the extracts as ingredients of functional foods for menopausal women susceptible to atherosclerosis. The present study was composed of four parts of experiments including four animal feeding experiments using ovariectomized rats, two cell culture ones using HepG2 cells and one sensory evaluation of test formulated products containing the extracts.
In the first part of the experiment, ethylacetate extract secondary to 80% ethanol extraction of defatted safflower seed and three types of safflower polyphenols were used in animal feeding experiment and the three purified polyphenols in cell culture experiment. Female Sprague-Dawley rats weighing 163.4±6.3 g were ovariectomized (Ovx) and fed either ethylacetate extract at a level of 1%(w/w) or three types of safflower polyphenolic compounds, i.e. lignans, flavones and serotonin derivatives at a level of 200 mg/kg diet containing 0.5%(w/w) cholesterol for four weeks. Sham and the Ovx control groups were fed the same diet without safflower components. Ovx groups had higher body weights but safflower seed extract or polyphenol did not affect uterus weights which were reduced by ovariectomy. Plasma GOT and GPT levels did not differ among the experimental groups. The plasma levels of total cholesterol were reduced in the four safflower groups by 20-30% compared with Ovx control. The plasma level of HDL-cholesterol was higher in the Ovx+ethylacetate extract group or appeared to be in the three Ovx+safflower polyphenolic groups than the Ovx control. The level of plasma triglyceride was also significantly lower in the Ovx+lignan group than in the Ovx control. The liver level of cholesterol was significantly reduced in the Ovx+ethylacetate extract group. Fecal excretion of cholesterol increased by safflower lignans and flavones whereas that of bile acid was not significantly changed by safflower polyphenols. Matairesinol and acacetin isolated from safflower seeds reduced cholesterol content in cultured HepG2 cells at a concentration of 0.01~0.1 uM and all three safflower polyphenols decreased triglyceride content at the concentration of 0.1 uM. These results suggest that safflower polyphenols have an effect on improving blood lipid status via increasing HDL-cholesterol formation and cholesterol excretion without significant uterotropic action in estrogen deficient animals.
In the second part of the experiment, a primary methanol extract (EL1), secondary butanol-soluble fraction(EL2) from Forsythia fruit(Forsythia viridissima L.) were used in animal feeding experiment and lignans, i.e. arctiin and matairesinol glucose in cell culture experiment. EL1 and EL2 were added at the levels of 2% (w/w) in diets and fed to ovariectomized rats, weighing 209.3±9.3 g for four weeks. Sham and the Ovx control groups were fed the same diet without Forsythia fruit components. The plasma level of triglyceride were significantly reduced in the EL1 group. The liver level of cholesterol and triglyceride was also fecal excretion of cholesterol and bile acid was not significantly changed by Forsythia fruit diet. Arctiin lowered cholesterol and triglyceride contents significantly in HepG2 cell cultured for three day at the concentration of 0.01, 0.1, 1 μM, but matairesinol glucose did not. These results indicates that the methanol extract (EL1) from forsythia fruit is effective on improving blood lipid status via decreased triglyceride without significant uterotropic action in estrogen deficient animals.
In the third part of experiment consisted of animal feeding only, defatted methanol extracts of the medicinal plants, Rubus coreanus Miq. (RC) and Atractylodes japonica Koidzumi (AJ) were added at the levels of 0.1, 0.5, or 2% (w/w) to high cholesterol diets and fed to ovariectomized rats, weighing 212.6±1.8 g for four weeks. Weight gains were lower in RC and AJ groups than the control group, but there were no changes in uterus weights. Serum levels of triglyceride decreased by 20～27% in the experimental groups fed 0.1% of each extract(0.1RC and 0.1AJ), compared with that of control(Ovx). Serum cholesterol levels were not changed in the RC groups but increased in the group fed 2% of the AJ extract. Liver levels of cholesterol and triglyceride were reduced in both the RC and AJ groups. Microscopic observation revealed that there were no morphological alterations in liver, lung, heart, spleen and kidney tissues of the experimental groups. Plasma levels of albumin, BUN, creatinine, sodium, potassium and phosphate in the RC and AJ groups were in normal ranges. Serum GOT and GPT activities were, however, higher in the 2AJ than Ovx group. These results suggest that the extracts of the Rubus coreanus Miq. and Atractylodes japonica Koidzumi at dietary levels as low as 0.1% are safely hypotriglyceridemic but not at the higher levels. In the fourth experiment, safflower seed products were formulated with the ethanol extract of the defatted seeds. The formulated product containing the ethanol extract, 10%, the extracts of RC, AJ and Eumonica ulmoides 2% each, ssangwha extract, 10%, glucose, 70% and lactose, 4% was included in the diet for six-month old ovariectomized rats (Ovx+Saf) at the level of 10% and fed for four weeks. Placebo product was also formulated without the ethanol extract of the defatted seeds and fed to another group of ovariectomized rats (Ovx+Placebo) at the same level. Serum total cholesterol and liver triglyceride and TBARS levels were decreased but serum HDL/-total cholesterol ratio and fecal excretions of cholesterol and bile acid were increased in the Ovx+Saf group compared with those of Ovx control and placebo groups. Serum levels of GOT, GPT, protein, albumin, BUN, creatinine, Na and K were not different among the experimental groups. No morphological alteration was found in the liver, lung, heart, spleen and kidney tissues of the Ovx+Saf group. The formulated products varying the levels of the extracts of the defatted safflower seed, RC, AJ and glucose were evaluated for their sensory preferences. The product containing the extract of the defatted safflower seed, 10%, the RC extract, <2% and the AJ extract, <1%.
It is concluded that the extracts of the defatted safflower seed, RC and AJ had the hypocholestrolemic and/or hypotriglyceridemic effects in ovariectomized rats and they can be utilized as functional food ingredients separately or in combination for improving body lipid status for the menopausal women.

목차 (Table of Contents)

• I. 서 론 1
• Ⅱ. 연구 배경 4
• 1. 에스트로겐과 지질대사 및 심혈관 조직 4
• 1-1. 콜레스테롤 및 중성지방 대사에 미치는 작용 4
• 1-2. 항산화작용 5
• 1-3 심혈관 조직 6
• 2. 에스트로겐 보충요법 6
• 3. 식물성 에스트로겐 7
• 4. 전통 약용 식물과 식물성 에스트로겐 12
• 4-1. 홍화 12
• 4-2. 연교 13
• 4-3. 복분자 14
• 4-4. 백출 14
• 5. 식물성 에스트로겐의 활용 15
• Ⅲ. 실험 재료 및 방법 18
• 1. 실험 설계 18
• 2. 실험 재료 및 추출물 제조 19
• 2-1. 식이성분 및 시약 19
• 2-2. 탈지 홍화씨 추출물 제조 및 성분 분리 19
• 2-3. 연교 추출물 제조 및 성분 분리 22
• 2-4. 복분자와 백출 추출물 제조 24
• 3. 약용 식물 추출물의 효능 평가를 위한 동물실험 26
• 3-1. 탈지 홍화씨 추출물 및 성분 식이실험 ( 1차 실험 ) 27
• 3-2. 연교 추출물 식이실험 ( 2차 실험 ) 29
• 3-3. 복분자와 백출 추출물 식이실험 ( 3차 실험 ) 30
• 3-4. 탈지 홍화씨 추출물 함유 복합제제 식이실험 ( 4차 실험 ) 31
• 4. 약용 식물 추출물의 효능 평가를 위한 세포 배양 실험 33
• 4-1. 탈지 홍화씨 폴리페놀 효능 ( 1차 실험 ) 33
• 4-2. 연교 폴리페놀 효능 ( 2차 실험) 33
• 5. 시료 34
• 5-1. 혈액 34
• 5-2. 간조직 및 기타 장기 34
• 5-3. 분변 34
• 5-4. HepG2 세포 34
• 6. 분석방법 35
• 6-1. 지질 분석 35
• 6-2. 혈액과 간조직 TBARS 측정 36
• 6-3. 분변 콜레스테롤과 담즙산 분석 36
• 6-4. 혈액 임상 화학 검사 36
• 6-5. 조직 형태 관찰 36
• 7. 통계처리 37
• Ⅳ. 결과 및 고찰 38
• 1. 1차 실험 38
• 1-1. 탈지 홍화씨 추출물 및 성분 식이실험 38
• 1-2. 탈지 홍화씨 폴리페놀에 의한 HepG2 세포내 지질 함량 변화 44
• 2. 2차 실험 46
• 2-1. 연교 추출물 식이실험 46
• 2-2. 연교 폴레페놀에 의한 HepG2 세포내 지질 함량 변화 51
• 3. 3차 실험 ; 복분자와 백출 추출물 식이실험 53
• 3-1. 체중 증가량과 식이 효율, 장기무게 53
• 3-2. 혈청 및 간 조직 지질 56
• 3-3. 혈청과 간의 TBARS 함량 59
• 3-4. 안전성 지표 61
• 3-5. 조직형태 관찰 61
• 4. 4차 실험 ; 탈지 홍화씨 추출물 함유 복합제제 식이실험 68
• 4-1. 체중 증가량, 식이 효율 및 장기무게 68
• 4-2 혈청, 간 및 분변 지질 70
• 4-3. 혈청과 간의 TBARS 함량 74
• 4-4. 안전성지표 76
• 4-5. 조직 형태 관찰 77
• 5. 탈지 홍화 씨 추출물 함유 복합제제 관능 평가 84
• Ⅴ. 요약 및 결론 86
• 참고문헌 89
• 부록 105