관절염 치료에 사용되고 있는 식물 추출물의 항염증 작용에 관한 연구 = Anti-inflammatory Activities of Plant Extracts used for Treatment of Arthritis

다국어 초록 (Multilingual Abstract)

ABSTRACT
Introduction: Among the various plants that exist in nature, some plant extracts have been used in areas, such as Chinese medicine, to treat arthritis.
The extracts of Ostericum koreanum, Curcumae rhizoma, Aralia continentalis, Achyanthes japonica, Paeonia lactiflora and Anemarrbena pbodeloides’s are known to be effective in treating arthritis in the arms.
In this study, the antioxidant and antimicrobial effects of these extracts are evaluated for associated inflammation with pathogenic bacteria
Methods : This paper presents research into the effects of these extracts as an anti-inflammatory reaction(inhibition of nitric oxide (NO) product) in lipopolysaccharide (LPS)-induced RAW 264.7 cells. The elements are extracted from the plant root with a solution of hot water and 95% ethanol.
1. Cytotoxicity of RAW 264.7 cells
To investigate the toxicity in RAW 264.7 cells of the elements extracted from various plants, different concentrations of 0, 10, 20, 40, 60, 80 and 100 μg/ml are used. After 24 hours the data is collected and examined with MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide, a tetrazole) assay. MTT assay was carried out as described by Mosmann( 1983), and Wilson (2000).
2. Inhibitory effects of NO production
Inhibitory effects of NO production on plant extracts were used LPS-induced RAW 264.7 cells. Cells were treated with various concentration (0, 10, 20, 40, 60, 80 and 100 μg/ml), and 1 ㎍/ml of LPS for 24 hr. NO production measured by the Griess reagent system as described in material and methods described by Yoon et al., (2007). Control sample indicates the LPS-stimulated cells. Statistically significant value compared with control group using SPSS 12.0 in term of t-test.
3. Antioxidative activity effects
DPPH (⍺-⍺-Diphenyl-β-picrylhydrazyl) radical scavenging activity of plant extracts were carried out as previously described by Blois (1958).
4. Anti-microbial activity effects on pathogenic bacteria
In this study, gram positive bacteria, Staphylococcus aureus KCCM 11593 and Staphylococcus pyrogenes ATCC 19615, and gram negative bacteria, Escherichia coli KCCM 1123, Pseudomonas aeruginosa ATCC 27853 and Salmonella typhimurium ATCC 11862 were used in this work. Antimicrobial activities effects of pathogenic bacteria were carried out by the methods of Lee (1999).
Results : The results of cytotoxicity of RAW 264.7 cells, anti-inflammatory activities, antioxidative activity effects and antimicrobial activity effects on pathogenic bacteria of plant extracts used for treatment of arthritis is given as follows.
1. Cytotoxicity of RAW 264.7 cells
The density examination of the elements of all plant extracts obtained by hot water provided good results that there were no toxic cells in any of the plant extracts. However, O. koreanum, A. continentalis, and A. pbodeloides’s extracts did not appear to be cytotoxic, when extracted with 95% ethanol.
However, C. rhizome appeared an 11.0% cell toxicity in density samples of 100 μg/ml. A. japonica has a 10.7% cell toxicity in 60 μg/ml, 13.1% in 80 μg/ml, and 13.9% in 100 μg/ml. P. lactiflora has an 8.4% cell toxicity in 80 μg/ml, and 16.4% in 100 μg/ml.
A mixed 1:1 extract of P. lactiflora and A. pbodeloides has a 6.5% cell toxicity in 20 μg/ml, 21.7% in 40 μg/ml, 3.71% in 60 μg/ml.
2. Inhibitory effects of NO production
For each of the plant extracts, NO product inhibition rate was measured for comparison to the control group.
The O. koreanum extracted by hot water and 95% ethanol showed NO product inhibition rate increases as the density rate increased. However, in hot water extract, it has appeared 16.0% in 80 ㎍/ml, but decreased in 100 ㎍/ml sample to 13.0%.
The C. rhizoma extracted by hot water and 95% ethanol showed NO product inhibition rate increases as the density rate increased. But, in hot water extract, it has a reduced result of 9.2%, as compared to 80 ㎍/ml in 100 ㎍/ml with 5.6%.
The A. continentalis extracted by hot water and 95% ethanol showed NO product inhibition rate increases as the density rate increased.
The A. japonica extracted by hot water has a 3.0% NO product inhibition rate in 10 ㎍/ml density, 4.0% in 20 ㎍/ml, 6.0% in 40 ㎍/ml, and 9.1% in 60 ㎍/ml. The NO product inhibition rate was 14.3.0% in 80 ㎍/ml, but has significantly decreased to -3.0% in 100 ㎍/ml.
The A. japonica extracted by 95% ethanol had NO product inhibition rate of 3.0% in 10 ㎍/ml density, 4.0% in 20 ㎍/ml, and 15.6% in 40 ㎍/ml, It has 50% in 60 ㎍/ml, 80 ㎍/ml and the NO product inhibition rate was 26.0% in 100 ㎍/ml.
The P. lactiflora extracted by hot water has NO product inhibition rates of 7.3% in 10 ㎍/ml density, 10.4% in 20 ㎍/ml, 10.8% in 40 ㎍/ml, and 13.0% in 60 ㎍/ml, 14.7% in 80 ㎍/ml. But, it has a reduced result of 5.2%, as compared to 80 ㎍/ml in 100 ㎍/ml with 9.5%.
The P. lactiflora extracted by 95% ethanol showed NO product inhibition rate increases as density rates increased.
The A. pbodeloides extracted by hot water and 95% ethanol had NO product inhibition rate increases along with the density rate.
A mixed 1:1 of P. lactiflora and A. pbodeloides extracted by hot water, had a NO product inhibition rate of 8.6% in 10 ㎍/ml density, 10.8% in 20 ㎍/ml, 19.1% in 40 ㎍/ml, 18.6% in 60 ㎍/ml, 16.5% in 80 ㎍/ml and 10.4% in 100 ㎍/ml. It show different results, as compared to extracts that were not mixed together.
A mixed 1:1 combination of P. lactiflora and A. pbodeloides, which was extracted by 95% ethanol, produced NO product inhibition rate increases, relative to the density rate.
3. Antioxidative activity effects
This section describes the antioxidative activity for the plant extracts, which were extracted by hot water. Experimental results show 81.97% in A. continentalis, 80.33% in P. lactiflora, 79.79% in O. koreanum, 79.22% in A. pbodeloides, and 77.45% in the hybrid combination of P. lactiflora and A. pbodeloides. It is very similar to results in the control group where the BHA was 82.9%, and the BHT was 83.1%. C. rhizoma has a 58.24%, and A. japonica has 36.16%, which are low rates in comparison to the control group.
This section describes the antioxidative activity for the plant extracts, which were extracted by 95% ethanol. Experimental data showed 81.03 % in O. koreanum, 76.75% in A. pbodeloides, 76.61% in P. lactiflora, 76.56% in mixed P. lactiflora and A. pbodeloides, and 72.44% in A. continentalis. This is a very similar rate, as compared to the control group. A. japonica has 41.50% and C. rhizoma has 39.20%, which are very low rates, as compared to the control group.
4. Anti-microbial activity effects on pathogenic bacteria
In the case of O. koreanum, for extracts using hot water, S. aureus bacteria started to appear as anti-microbial activity in 700 ㎍/ml, and it has 35.9% anti-microbial activity in 1,500 ㎍/ml. S. pyrogenes bacteria has 48.0% anti-microbial activity in 1,500 ㎍/ml. Other bacteria do not have any anti-microbial activity.
In the case of O. koreanum, extracts using 95% ethanol, S. aureus bacteria has 93.3% anti-microbial activity in 700 ㎍/ml, S. pyrogenes bacteria has 99.1% anti-microbial activity in 700 ㎍/ml.
In the case of C. rhizoma, extracted by hot water, S. pyrogenes bacteria has 18.2% anti-microbial activity in 1,500 ㎍/ml, and there is no anti-microbial activity in other bacteria.
In the case of C. rhizoma extracted by 95% ethanol, S. aureus bacteria has 95%, and P. aeruginosa bacteria has 100% anti-microbial activity in 1,500 ㎍/ml. In other bacteria, S. pyrogenes bacteria has 27.3%, E. coli bacteria has 51.39%, and S. typhimurium bacteria has 86.6% in 1500 ㎍/ml.
In case of A. continentalis extracted by hot water, S. pyrogenes bacteria has 87.9% anti-microbial activity in 700 ㎍/ml, 96.8% in 1,500 ㎍/ml. But E. coli bacteria has 4.3%, and P. aeruginosa bacteria has 18.2% anti-microbial activity in 1,500 ㎍/ml. In other bacteria, there isn’t any anti-microbial activity.
In A. continentalis extracted by 95% ethanol, S. aureus bacteria has 97.0%, and S. typhimurium bacteria has 88.0% in 100 ㎍/ml. S. pyrogenes has 60.6%, E. coli has 8.2%, and P. aeruginosa has 30.1% in 1,500 ㎍/ml.
In A. japonica, S. pyrogenes bacteria has 27.9% in hot water extraction and 1,500 ㎍/ml, and 48.7% in 95% ethanol and 1,500 ㎍/ml. There is no anti-microbial activity in other bacteria.
In the case of P. lactiflora extracted by hot water, S. pyrogenes bacteria has 90.9% anti-microbial activity in 700 ㎍/ml, 91.7% in 1,500 ㎍/ml, and no anti-microbial activity in other bacteria.
In P. lactiflora extracted by 95% ethanol, S. aureus bateria has 87.0% in 1,500 ㎍/ml, S. pyrogenes has 84.7% in 300 ㎍/ml, and 100% in 1,500 ㎍/ml. E. coli has 36.5% in 1,500 ㎍/ml, P. aeruginosa has 32.6% in 1,500 ㎍/ml, and S. typhimurium bacteria doesn’t have any anti-microbial activity in 1,500 ㎍/ml.
In A. pbodeloides extracted by hot water, S. pyrogenes bacteria has 43.0% anti-microbial activity in 1,500 ㎍/ml and no anti-microbial activity in other bacteria.
In A. pbodeloides extracted by 95% ethanol, S. aureus bateria has 13.4% in 1,500 ㎍/ml and S. pyrogenes has 77.1% in 700 ㎍/ml, 89.0% in 1,500 ㎍/ml. Other bacteria don’t have any anti-microbial activity.
In case of the 1:1 mixture of P. lactiflora and A. pbodeloides extracted by hot water, S. pyrogenes bacteria has 14.2% anti-microbial activity in only 1,500 ㎍/ml, with no anti-microbial activity in other bacteria.
In a mixture of P. lactiflora and A. pbodeloides extracted by 95% ethanol, S. aureus has 41.6%, S. pyrogenes has 92.2%, and E. coli has 7.8% in 1,500 ㎍/ml. There was no anti-microbial activity in other bacteria.

목차 (Table of Contents)

• ABSTRACT III
• I. 서 론 1
• II. 이론적 배경 5
• III. 재료 및 방법 30
• 1. 실험재료 30
• 1) 사용한 식물 30
• 2. 추출물의 조제 30
• 3. 세포독성 측정 31
• 4. NO 생성 제어효과 측정 32
• 5. 항산화 활성 측정 33
• 6. 병원성 미생물에 대한 항균활성 측정 33
• 7. 통계분석 34
• IV. 결과 및 고찰 35
• 1. RAW 264.7 세포 35
• 2. 식물 추출물들의 RAW 264.7 세포에 대한 세포독성 36
• 3. 염증유발 물질인 NO 생성 저해효과 40
• 1) 강활 (O. koreanum) 추출물 42
• 2) 강황 (C. rhizoma) 추출물 44
• 3) 독활 (A. continentalis) 추출물 45
• 4) 우슬 (A. japonica) 추출물 48
• 5) 작약 (P. lactiflora) 추출물 50
• 6) 지모 (A. aspbodeloides) 추출물 52
• 7) 작약 (P. lactiflora)과 지모 (A. aspbodeloides)의 혼합
• 추출물 52
• 3. 항산화 활성효과 57
• 4. 병원성 세균에 대한 항균활성 효과 61
• 1) 강활 (O. koreanum) 추출물의 항균활성 효과 61
• 2) 강황 (C. rhizoma) 추출물의 항균활성 효과 66
• 3) 독활 (A. continentalis) 추출물의 항균활성 효과 71
• 4) 우슬 (A. japonica) 추출물의 항균활성 효과 75
• 5) 작약 (P. lactiflora) 추출물의 항균활성 효과 78
• 6) 지모 (A. aspbodeloides) 추출물의 항균활성 효과 82
• 7) 작약 (P. lactiflora)과 지모 (A. aspbodeloides)의 혼합
• 추출물의 항균활성 효과 85
• V. 요약 90
• 참고문헌 97
• List of Figures 120
• List of Tables 124