화장품 신소재 개발을 위한 조직배양 노니(Morinda citrifolia L.) 부정근의 효능평가와 유효성분 분석 = Efficacy evaluation and analysis of active component in tissue cultured Noni (Morinda citrifolia L.) adventitious roots for the development of new cosmetic materials

다국어 초록 (Multilingual Abstract)

This study evaluated anti-oxidant, whitening, wrinkles effects, and anti-allergic effects of the noni (Morinda citrifolia L.) adventitious roots, selected an indicator material showing a great anti-allergic effects, and then established qualitative and quantitative methods for purification and structural analyses of the active ingredient. Also, several different extraction and drying methods were compared to see change in rubiadin, anthraquinone, phenolic, flavonoid, and extraction yield, and to identify effective processing.



1. Evaluation of biological activity

The toxicity on the extracts of tissue cultured noni adventitious roots, were evaluated, and their effects on DPPH scavenging, SOD-like, and protection against H2O2 induced cell damage were identified and proved to be slight. As a result of tyrosinase inhibition, inhibition of melanin in B16F1 melanoma, and western blotting of tyrosinase, TRP-1, and TRP-2 expression, the effect of whitening activity was minor. The activity of elastase inhibition, stimulation of collagen, and procollagen type-Ⅰ synthesis was little which means that the effect on wrinkles was negligible.
However, when the noni adventitious roots were evaluated on anti-allergic, crude extracts showed excellent effectiveness on histamine releasing inhibition of 85% at 100 μg·mL-1. Thus, liquid-liquid extractions were conducted using several organic solvents and each fraction was evaluated on histamine releasing and TNF-α synthesis inhibition. Among them, hexane and methylene chloride extracts were more effective than others, in which the hexane fraction showed about 54% efficacy at 25 μg·mL-1. In the results of the effect of lethality inhibition through systemic anaphylactic reaction on tissue cultured. lethality by systemic anaphylactic reaction decreased by almost 36% in the groups that noni adventitious roots and stem cell medicated were orally administered. Also, as a result of evaluated effect of histamine releasing inhibition using a rat visceral mast cell, it was controlled effectively when treated with 1 mg·mL-1.





2. Selection of target compound

To select target compounds with anti-allergic activity in noni adventitious, the extracts were partitioned with solvents. Active ingredients obtained from hexane and methylene chloride layers were a fraction with preparative LC. Target compound from HPLC was identified by GC/MS/MS, LC/MS-IT-TOF, and UV/VIS spectrophotometer. From these results, rubiadin was selected as a target compound.
In addition, the structural analysis was conducted through extraction and purification of rubiadin in noni adventitious roots to establish qualitative analysis. Noni adventitious roots were extracted with chloroform : ethyl acetate mixture (100%, 100:1, v/v). The extract was developed by preparative TLC with chloroform : ethyl acetate (3:1, v/v), a band Rf 0.68 were collected, and. further purification was conducted to get rubiadin by preparative LC. Purified rubiadin was identified by LC/MS/MS, LC/MS-IT-TOF, UV/VIS spectrophotometer, FT-IR, and NMR analysis.


3. Efficiency of extraction and dry methods

To evaluated optimum extraction condition on rubiadin, extraction of noni adventitious roots was performed with different solvent types, ratio of water to methanol (20%, 40%, 60%, 80%, and 100% water), extraction time, and extraction method. In contrast, a reverse-phase HPLC assay method was developed to determine rubiadin content in noni adventitious roots in which HPLC equipped with a C-18 column using a gradient solvent system of methanol and water and UV detector at 280 nm was used.
As a result, roots contained 0.02% of rubiadin but no rubiadin was detected in other plant part. Rubiadin, anthraquinones, phenolic, flavonoid content and extraction yields in the adventitious roots were higher than theose in the ex vitro roots.
The extraction efficiency using different types of solvents were increased in order of methanol (0.08%) > ethanol (0.05%) > acetonitrile (0.03%) > acetone (0.02%) and methylene chloride (0.02%). Rubiadin extraction using different solvents showed that 1 hour of ultrasonic extraction was effective in order of 60 % methanol (0.21%) > 80% methanol (0.13%) > 100% methanol (0.07%), 40% methanol (0.07%) and 2 hours of reflux extraction was effective in order of 60% methanol (0.21%) > 40% methanol (0.17%) > 80% methanol (0.14%).
To compare the extraction efficiency of active ingredient by the extraction methods and extraction time for noni adventitious roots were extracted by ultrasonic extraction, shaking extraction, reflux extraction, homogenizer extraction, high pressure extraction, and soaking extraction. The order of extraction efficiency of rubiadin content was homogenizer extraction (2 h) > ultrasonic extraction (8 h) > shaking extraction (24 h) > high pressure extraction (24 h) > soaking extraction (6 h) > reflux extraction (2 h). The highest phenolic content was found in the extraction from ultrasonic extraction and anthraquinone content was highest in the reflux extraction. But extraction yield was found to be insignificant differences according to the extraction methods
Two-time extraction and 1:100 were promising for cost-effective method. To find out the best drying methods, noni adventitious roots were dried using freeze dry, oven dry, and shade dry method. Rubiadin, anthraquinone, and extraction yield were highest in the freeze dry method.


4. Conclusion

Noni adventitious roots from bioreactor culture conditions were excellent in anti-allergic activity. Rubiadin, a target compound was isolated from noni adventitious roots and its structure was identified by spectral data. To evaluate the effective extraction condition of the active ingredient, extraction process of noni adventitious roots was performed with various solvents, apparatus, and drying methods. The results suggested that the noni adventitious roots can be used for valuable resource.

목차 (Table of Contents)

• Ⅰ. 서언 1
• Ⅱ. 연구사 5
• 1. 부정근 조직배양 5
• 2. 생리활성물질의 효능평가 8
• 3. 유효성분 분리정제 및 구조분석 10
• 4. 추출용매 및 추출방법, 가공방법에 따른 유효성분의 추출효율에 미치는 영향 14
• 4.1. 추출용매 14
• 4.2. 추출방법 15
• 4.3. 가공방법 18
• III.재료 및 방법. 19
• 1.노니 부정근 조직배양 19
• 1.1.식물재료 19
• 1.2.노니 세포 유도 및 증식 20
• 1.3.노니 부정근의 유도 및 증식 21
• 1.4.조직배양 노니 부정근의 생물반응기 배양 21
• 2.노니 부정근의 효능평가 22
• 2.1.노니 추출물의 항산화 효과 22
• 2.1.1.HDF를 이용한 세포독성 측정 22
• 2.1.2.DPPH를 이용한 전자공여능 측정 23
• 2.1.3.SOD 유사활성 측정 23
• 2.1.4.과산화물에 의한 세포 손상 억제 측정 24
• 2.2.노니 추출물의 미백 효과 25
• 2.2.1.Mushroom tyrosinase를 이용한 melanin 생성 억제 효과 25
• 2.2.2.B16F1 melanoma를 이용한 melanin 생성 억제 효과 25
• 2.2.3.B16F1 melanoma 내 멜라닌 생합성관련 효소 억제 효과 26
• 2.3.노니 추출물의 주름개선 효과 27
• 2.3.1.PPE를 이용한 주름개선 효과 27
• 2.3.2.인체 섬유아세포를 통한 collagen 생성 촉진 효과 28
• 2.3.3.인체 섬유아세포 유래 procollagen type-Ⅰ 생성 촉진 효과 28
• 2.4.노니 추출물의 항알러지 효과 30
• 2.4.1.노니 조추출물에 대한 rat RBL-2H3를 이용한 histamine 발현 억제 효과 30
• 2.4.2.노니 용매 분획물에 대한 항알러지 효과 31
• ①노니 부정근 용매 분획물 제조 31
• ②HDF를 이용한 세포 독성 측정 32
• ③Rat RBL-2H3를 이용한 histamine 발현 억제 효과 32
• ④Rat RBL-2H3를 이용한 TNF-α 발현 억제 효과 32
• ⑤Rat RBL-2H3를 이용한 interleukin(IL-4) 발현 억제 효과 33
• ⑥HMC-1을 이용한 caspase-1 발현 억제 효과 33
• ⑦HMC-1을 이용한 pro-caspase-1과 caspase-1 발현 억제 효과 34
• 2.4.3.노니 조직배양체 조추출물에 대한 항알러지 동물실험 34
• ①Systemic anaphylactic reaction을 통한 치사율 억제 효과 34
• ②Rat 복강 비만세포로부터의 histamine 유리 억제 효과 35
• 3.항알러지 성분의 정제 및 구조분석을 통한 지표물질의 선정 36
• 3.1.항알러지 성분의 추출 및 분리, 분석 36
• 3.1.1.조추출물에 대한 용매별 분획 추출 및 분리 36
• 3.1.2.분취 화합물 분석 37
• 3.2.노니 부정근 내 rubiadin 분리정제 및 구조분석 39
• 3.2.1.노니 부정근 추출 및 정제 39
• 3.2.2.Rubidin 구조분석 40
• 3.3.지표물질선정 42
• 4.추출조건 및 건조방법에 따른 유효성분의 추출효율에 미치는 영향 43
• 4.1.분석조건 확립 43
• 4.1.1.표준용액 제조 43
• 4.1.2.Rubiadin의 HPLC 분석조건 43
• 4.1.3.고형분 함량 측정 43
• 4.1.4.Anthraquinone 함량 측정 44
• 4.1.5.플라보노이드 함량 측정 44
• 4.1.6.총 페놀 함량 측정 45
• 4.2.기외 식물체 및 조직배양체 유효성분 함량 46
• 4.2.1.기외 식물체 부위별 유효성분 함량 46
• 4.2.2.기외 식물체 및 조직배양된 부정근의 유효성분 함량 46
• 4.3.추출조건에 따른 유효성분의 함량에 미치는 영향 46
• 4.3.1.추출용매 46
• 4.3.2.추출용매 혼합 비율 47
• 4.3.3.추출시간 및 추출방법 47
• 4.3.4.추출용매 비율 49
• 4.3.5.추출 횟수 49
• 4.4.건조방법에 따른 유효성분의 함량에 미치는 영향 49
• IV.결과 및 고찰 50
• 1.노니 부정근 효능평가 50
• 1.1.노니 추출물의 항산화 효과 51
• 1.2.노니 추출물의 미백 효과 58
• 1.3.노니 추출물의 주름개선 효과 64
• 1.4.노니 추출물의 항알러지 효과 69
• 1.4.1.노니 조추출물 및 분획물의 항알러지 효과 69
• 1.4.2.노니 조직배양체 조추출물의 항알러지 동물 실험. 81
• 2.항알러지 성분의 정제 및 구조분석 85
• 2.1항알러지 성분 추출 및 분리, 분석 85
• 2.2.노니 부정근 내 rubiadin 분리정제 및 구조분석 94
• 2.2.1노니 부정근 추출 및 정제 94
• 2.2.2.Rubiadin 구조분석 100
• 2.3.노니 부정근의 지표물질 선정 107
• 3.추출조건 및 건조방법에 따른 유효성분의 추출효율에 미치는 영향 112
• 3.1.분석조건 확립 112
• 3.2기외 식물체 및 조직배양체 유효성분 함량 114
• 3.2.1.기외 식물체 부위별 유효성분 함량 114
• 3.2.2.기외 식물체 및 조직배양체 유효성분 함량 118
• 3.3추출조건에 따른 유효성분의 함량에 미치는 영향 122
• 3.3.1.추출용매에 따른 유효성분 함량에 미치는 영향 122
• 3.3.2.추출용매 혼합 비율에 따른 유효성분 함량에 미치는 영향 127
• 3.3.3.추출시간 및 추출방법에 따른 유효성분의 함량에 미치는 영향 131
• 3.3.4.추출용매 비율에 따른 유효성분 함량에 미치는 영향 143
• 3.3.5.추출 횟수에 따른 유효성분 함량에 미치는 영향 146
• 4.건조방법에 따른 유효성분 함량에 미치는 영향 149
• Ⅴ 적요 153
• Ⅵ.인용문헌 156