Chemical constituents of armoracia rusticana and rosa multiflora and their neuroprotective activities

다국어 초록 (Multilingual Abstract)

As a part of our ongoing search for bioactive constituents from Korean medicinal plants, the roots of A. rusticana and the twigs of R. multiflora were investigated. The investigation resulted in the isolation and characterization of 39 compounds from the roots of A. rusticana and 28 compounds from the twigs of R. multiflora. The structures of the isolated compounds were elucidated through spectroscopic analysis, including NMR (1H and 13C NMR, 1H-1H COSY, HSQC, HMBC, and NOESY), spectrometric analysis (LC-MS and HRESIMS), and chemical methods. Moreover, the configurational assignment was conducted using experimental and calculated ECD and LC-MS analysis. The isolated compounds from the roots of A. rusticana were identified as 2-allyl-8-hydroxy-3-thioxohexahydro-1H-pyrrolo[1,2-c]imidazole-1-one (A-1), 2-allyl-8-hydroxytetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (A-2), 8-hydroxy-2-phenylethyl-3-thioxohexahydro-1H-pyrrolo[1,2-c]imidazole-1-one (A-3), 8-hydroxy-2-phenylethyltetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (A-4), horsethiohydantoin (A-5), (S)-2-phenylethyltetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (A-6), horseradiurea (A-7), (S)-(-)-spirobrassinin (A-8), phenethyl cyanide (A-9), N-(2-phenylethyl)urea (A-10), horseramide A (A-11), 4-(methylsulfinyl)butanenitrile (A-12), 6-(methylsulfinyl)hexanenitrile (A-13), 7-(methylsulfinyl)heptanenitrile (A-14), 8-(methylsulfinyl)octanenitrile (A-15), 9-(methylsulfinyl)nonanenitrile (A-16), 10-(methylsulfinyl)decanenitrile (A-17), 9-(methylsulfinyl)nonanoic acid (A-18), 8-(methylsulfonyl)octanenitrile (A-19), 9-(methylsulfonyl)nonanenitrile (A-20), kaempferol (A-21), kaempferol 3-O-β-D-glucopyranoside (A-22), kaempferol 3-O-β-D-galactopyranoside (A-23), kaempferol 3-O-α-D-arabinopyranoside (A-24), kaempferol 3-O-β-D-xylopyranoside (A-25), horseradiside A-C (A-26 ~ A-28), kaempferol 3-O-β-D-xylopyranoside-7-β-D-glucopyranoside (A-29), kaempferol 3-O-β-D-xylopyranosyl-(1′′′→2′′)-β-D-glucopyranoside (A-30), kaempferol 3-O-β-D-xylopyranosyl-(1′′′→2′′)-β-D-galactopyranoside (A-31), kaempferol 3-O-β-D-apiofuranosyl-(1′′′→2′′)-β-D-glucopyranoside (A-32), nicotiflorin (A-33), kaempferol 3-O-β-D-glucopyranoside-4′-β-D-glucopyranoside (A-34), (7S,8R)-3′-demethyl-dehydrodiconiferyl alcohol-3′-O-β-D-glucopyranoside (A-35), horseradiside D (A-36), lariciresinol-4′-O-β-D-glucoside (A-37), lariciresinol-4-O-β-D-glucoside (A-38), and (+)-lariciresinol-4,4′-O-bis-β-D-glucopyranoside (A-39). Consequently, compounds A-1 ~ A-7, A-11, A-26 ~ A-28, and A-36 were confirmed as new compounds. Furthermore, the structures of the isolated compounds from the twigs of R. multiflora were identified as jillenidin A-C (R-1 ~ R-3), procyanidin B6 (R-4), procyanidin B5 (R-5), rosaside A (R-8), catechin (R-9), (+)-catechin 7-O-β-D-glucopyranoside (R-10), (+)-catechin 5-O-β-D-glucopyranoside (R-11), (+)-catechin 4′-O-β-D-glucopyranoside (R-12), rosaside B (R-13), citrusin A (R-14), 4-[(1R,2S)-1,3-dihydroxy-2-[4-[(1E)-3-hydroxy-1-propen-1-yl]-2-methoxyphenoxy]propyl]-2-methoxyphenyl β-D-glucopyranoside (R-15), rosaside C (R-16), methyl 3,4-dihydroxy-5-[[6-O-(3,4,5-trihydroxybenzoyl)-β-D-glucopyranosyl]oxy]benzoate (R-17), methyl gallate 3-O-β-D-glucopyranoside (R-18), rosaside D (R-19), 2,4,6-trimethoxyphenyl β-D-glucopyranoside (R-20), leonuriside A (R-21), betulin (R-22), lupeol (R-23), pomolic acid (R-24), corosolic acid (R-25), maslinic acid (R-26), arjunolic acid (R-27), and hederagenin (R-28).
All of the isolated compounds were evaluated for their potential neurotrophic activity through induction of nerve growth factor (NGF) in C6 glioma cell lines and their anti-neuroinflammatory activity based on the measurement of inhibition levels of nitric oxide (NO) production in lipopolysaccharide (LPS)-activated microglia BV-2 cells. Compounds A-1b, A-2a, and A-7 were mainly exhibited potent neurotrophic activities (stimulation levels: 153.59 ± 5.44, 141.99 ± 5.21, and 157.06 ± 2.23%, respectively) and compounds A-21 ~ A-25, A-27, A-31, A-32, and A-35 were showed powerful NGF secretion with the stimulation levels of 167.08 ± 3.53, 143.56 ± 1.94, 154.01 ± 7.84, 159.45 ± 1.41, 153.36 ± 17.30, 129.09 ± 13.73, 176.96 ± 2.58, 174.48 ± 2.10, and 170.03 ± 0.64%, respectively. Furthermore, compound A-7 exhibited potent NO inhibition effect with an IC50 value of 19.83 μM. Besides, R-8, R-13, and R-21 exhibited moderate anti-neuroinflammatory activities with IC50 values of 51.52, 50.58, and 44.32 μM, respectively.

국문 초록 (Abstract)

국내 자생하는 천연식물로부터 새로운 퇴행성 신경질환 치료제의 개발을 위한 노력의 일환으로 수종의 국내 자생 식물을 채취하여 신경보호 prescreening 방법을 사용하여 Nerve Growth Factor (NGF, 신경성장인자) 분비유도활성과 Nitric Oxide (NO, 신경염증 유발인자) 생성억제활성을 평가하였다. 그 결과 Armoracia rusticana (겨자무)의 뿌리와 Rosa multiflora (찔레나무)의 가지에서 유의한 활성이 확인되었고, 두 식물들로부터 신경영양인자 분비유도 및 신경염증
억제활성에 대한 성분 연구를 수행하였다.
먼저, A. rusticana (겨자무) 뿌리의 신경보호 성분 연구를 수행하였다. 그 결과 80% 메틸알콜 추출물로부터 얻어진 분획물 중 클로로포름 분획에서 6종의 새로운 thiohydantoin 성분과 5종의 새로운 hydantoin 성분, 1종의 새로운 hydantoinurea 성분, 그리고 1종의 새로운 methyl sulfinyl derivative 성분을 포함한 총 20종의 물질이 분리되었다. 이와함께 에틸아세테이트 및 n-부틸알콜 분획에서 3종의 새로운 kaempferol 배당체성분과 1종의 새로운 lignin 배당체를 포함한 11종의 성분이 분리되었다. 분리된 39 종의 성분들은 그들의 이화학적 성상 및 기기분석결과를 통해 그 구조를 2-allyl-8-hydroxy-3-thioxohexahydro-1H-pyrrolo[1,2-c]imidazole-1-one (A-1), 2-allyl-8-hydroxytetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (A-2), 8-hydroxy-2-phenylethyl-3-thioxohexahydro-1H-pyrrolo[1,2-c]imidazole-1-one (A-3), 8-hydroxy2-phenylethyltetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (A4), horsethiohydantoin (A-5), (S)-2-phenylethyltetrahydro-1Hpyrrolo[1,2-c]imidazole-1,3(2H)-dione (A-6), horseradiurea (A-7), (S)-(-)-spirobrassinin (A-8), phenethyl cyanide (A-9), N-(2-phenylethyl)urea (A-10), horseramide A (A-11), 4-(methylsulfinyl)butanenitrile (A-12), 6-(methylsulfinyl)hexanenitrile (A13), 7-(methylsulfinyl)heptanenitrile (A-14), 8-(methylsulfinyl)octanenitrile (A-15), 9-(methylsulfinyl)nonanenitrile (A-16), 10-(methylsulfinyl)decanenitrile (A-17), 9-(methylsulfinyl)nonanoic acid (A18), 8-(methylsulfonyl)octanenitrile (A-19), 9-(methylsulfonyl)nonanenitrile (A-20), kaempferol (A-21), kaempferol 3-Oβ-D-glucopyranoside (A-22), kaempferol 3-O-β-D-galactopyranoside
(A-23), kaempferol 3-O-α-D-arabinopyranoside (A-24), kaempferol 3-O-β-D-xylopyranoside (A-25), horseradiside A-C (A-26 ~ A-28),kaempferol 3-O-β-D-xylopyranoside-7-β-D-glucopyranoside (A-29),kaempferol 3-O-β-D-xylopyranosyl-(1′′′→2′′)-β-D-glucopyranoside (A-30), kaempferol 3-O-β-D-xylopyranosyl-(1′′′→2′′)-β-Dgalactopyranoside (A-31), kaempferol 3-O-β-D-apiofuranosyl-(1′′′→2′′)-β-D-glucopyranoside (A-32), nicotiflorin (A-33), kaempferol 3-O-β-Dglucopyranoside-4′-β-D-glucopyranoside (A-34), (7S,8R)-3′-demethyldehydrodiconiferyl alcohol-3′-O-β-D-glucopyranoside (A-35),horseradiside D (A-36), lariciresinol-4′-O-β-D-glucoside (A-37),lariciresinol-4-O-β-D-glucoside (A-38), 그리고 (+)-lariciresinol-4,4′-O-bis-β-D-glucopyranoside (A-39)로 구조규명 하였고, 위 성분들에 대한 신 경영양인자 분비활성과 신경염증인자 분비억제 활성을 평가하였다. 그 결과 화합물 A-1b, A-2a, 그리고 A-7에서 매우 강한 NGF 분비유도활성이 확인되었다. (stimulation levels: 153.59 ± 5.44, 141.99 ± 5.21, 그리고 157.06 ±2.23%). 이와함께 화합물 A-21 ~ A-25, A-27, A-31, A-32, 그리고 A-35에서 매우 강한 NGF 분비유도활성이 확인되었다 (stimulation levels: 129.09 ± 13.73- 176.96 ± 2.58%). 흥미롭게도, 화합물 A-7에서는 매우 강한 NO생성 억제활성 또한 확인할 수 있었다 (IC50 19.83 μM).
다음으로, R. multiflora (찔레나무)의 가지에 대한 신경보호 성분 연구를 수행하였다. 그 결과, 80% 메틸알콜 추출물로부터 얻어진 분획물 중 에틸아세테이트, n부틸알콜, 그리고 클로로포름 분획에서 5종의 새로운 biflavonoid 성분들과 1종의 새로운 flavanol 배당체, 1종의 새로운 lignin 배당체, 그리고 2종의 새로운 phenolic 배당체를 포함한 총 28종의 물질이 분리되었다. 분리된 28 종의 성분들은 그들의 이화학적 성상 및 기기분석결과를 통해 그 구조를 jillenidin A-C (R-1~ R-3), procyanidin B6 (R-4), procyanidin B5 (R-5), rosaside A (R-8), catechin (R-9), (+)-catechin 7-O-β-D-glucopyranoside (R-10), (+)-catechin 5-O-β-D-glucopyranoside (R-11), (+)-catechin 4′-O-β-Dglucopyranoside (R-12), rosaside B (R-13), citrusin A (R-14), 4-[(1R,2S)-1,3-dihydroxy-2-[4-[(1E)-3-hydroxy-1-propen-1-yl]-2-methoxyphenoxy]propyl]-2-methoxyphenyl β-D-glucopyranoside (R-15),rosaside C (R-16), methyl 3,4-dihydroxy-5-[[6-O-(3,4,5-trihydroxybenzoyl)-β-D-glucopyranosyl]oxy]benzoate (R-17), methylgallate 3-O-β-D-glucopyranoside (R-18), rosaside D (R-19), 2,4,6-trimethoxyphenyl β-D-glucopyranoside (R-20), leonuriside A (R-21), betulin (R-22), lupeol (R-23), pomolic acid (R-24), corosolic acid (R-25), maslinic acid (R-26), arjunolic acid (R-27), 그리고 hederagenin (R-28)로 구조규명 하였고, 위 성분들에 대한 신경영양인자 분비활성과 신경염증인자 분비억제활성을 평가하였다. 그 결과 화합물 R-8, R-13, 그리고 R-21에서 NO생성 억제를 통한 약하지만 유의한 항신경염증활성이 관찰되었다 (51.52, 50.58, 그리고 44.32 μM).
이상의 연구를 통하여 A. rusticana (겨자무)의 뿌리와 R. multiflora (찔레나무)의 가지로부터 총 26 종의 새로운 화합물 (6종의 thiohydantoin, 5종의 hydantoin, 1종의 hydantoinurea, 1종의 methyl sulfinyl derivative, 4종의 flavonoid 배당체, 2종의 lignan 배당체, 5종의 biflavonoid, 1종의 flavanol 배당체, 1종의 lignin 배당체 그리고 2종의 phenolic 배당체 성분)을 포함한 총 67종의 성분을 분리하여 그 구조를 규명하였다. 구조규명을 완려한 후에는 신경영양인자 (NGF) 분비유도활성과 신경염증인자 (NO) 생성억제활성을 평가하였다. 분리된 물질 중 16 종의 물질에서 유의성 있는 신경영양인자 (NGF) 분비유도활성 또는 신경염증인자 (NO) 생성억제활성을 확인하였고, 특히 화합물 A-7 (horseradiurea)는 신경영양인자(NGF) 분비유도활성과 신경염증인자 (NO) 생성억제활성 모두에서 매우 강한 활성을 나타내었다. 두 신경보활성평가의 결과는, 화합물 A-7이 향후 신경보호활성의 약물을 개발함에 있어 매우 유망한 후보물질로의 가능성을 나타낸것으로 판단된다.

목차 (Table of Contents)

• Chapter 1. Introduction 1
• Chapter 2. Materials and Methods 10
• 1. Materials 10
• 1.1. Plant materials 10
• 1.1.1. A. rusticana 10
• 1.1.2. R. multiflora 10
• 2. Biological Evaluation 14
• 2.1. Cell lines 14
• 2.2. Methods 14
• 2.2.1. NGF and Cell viability assays 14
• 2.2.2. NO Production and Cell Viability Assay 15
• 3. Isolation and purification 17
• 3.1. A. rusticana 17
• 3.2. R. multiflora 56
• Chapter 3. Results 88
• 1. Structure elucidation of compounds isolated from A. rusticana 88
• 1.1. The structure of compound A 1 88
• 1.2. The structure of compound A 2 95
• 1.3. The structure of compound A 3 101
• 1.4. The structure of compound A 4 107
• 1.5. The structure of compound A 5 113
• 1.6. The structure of compound A 6 119
• 1.7. The structure of compound A 7 124
• 1.8. The structure of compound A 11 129
• 1.9. The structure of compound A 26 134
• 1.10. The structure of compound A 27 139
• 1.11. The structure of compound A 28 144
• 1.12. The structure of compound A 36 149
• 1.13. The structures of compounds A 8 ~ A 10, A 12 ~ A 25, A 29 ~ A 35, and A 37 ~ A 39 159
• 2. Structure elucidation of compounds isolated from R. multiflora 160
• 2.1. The structure of compound R 1 160
• 2.2. The structure of compound R 2 169
• 2.3. The structure of compound R 3 176
• 2.4. The structure of compound R 6 183
• 2.5. The structure of compound R 7 191
• 2.6. The structure of compound R 8 198
• 2.7. The structure of compound R 13 206
• 2.8. The structure of compound R 16 217
• 2.9. The structure of compound R 19 222
• 2.10. The structures of compounds R 4, R 5, R 9 ~ R 12, R 14, R 15, R 17, R 18, R 20, and R 21 229
• 3. Neuroprotective activities of compounds 235
• Chapter 4. Conclusion 245
• References 249
• 논문요약 262