องคประกอบทางเคมีและฤทธิ์ทางชีวภาพของตนสิรนิ ธรวัลลีและตนเปลาแพะ
นางสาวศิริวรรณ อธิคมกุลชัย
วิทยานิพนธนี้เปนสวนหนึ่งของการศึกษาตามหลักสูตรปริญญาวิทยาศาสตรดุษฎีบณ
ั ฑิต
สาขาวิชาเภสัชเคมีและผลิตภัณฑธรรมชาติ
คณะเภสัชศาสตร จุฬาลงกรณมหาวิทยาลัย
ปการศึกษา 2547
ISBN : 974-17-5964-9
ลิขสิทธิ์ของจุฬาลงกรณมหาวิทยาลัย
CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF BAUHINIA
SIRINDHORNIAE AND CROTON HUTCHINSONIANUS
Miss Sirivan Athikomkulchai
A Dissertation Submitted in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy in Pharmaceutical Chemistry and Natural Products
Faculty of Pharmaceutical Sciences
Chulalongkorn University
Academic Year 2004
ISBN : 974-17-5964-9
Thesis Title
CHEMICAL CONSTITUENTS AND BIOLOGICAL
ACTIVITIES OF BAUHINIA SIRINDHORNIAE AND
CROTON HUTCHINSONIANUS
By
Miss Sirivan Athikomkulchai
Field of Study
Pharmaceutical Chemistry and Natural Products
Thesis Advisor
Associate Professor Nijsiri Ruangrungsi, Ph.D.
Thesis Co-Advisor
Professor Somsak Ruchirawat, Ph.D.
Thesis Co-Advisor
Associate Professor Nongluksna Sriubolmas, Ph.D.
Accepted by the Faculty of Pharmaceutical Sciences, Chulalongkorn
University in Partial Fulfillment of the Requirements for the Doctor’s Degree
………………………………Dean of the Faculty of Pharmaceutical Sciences
(Associate Professor Boonyong Tantisira, Ph.D.)
THESIS COMMITTEE
……………………………………………….. Chairman
(Associate Professor Sumphan Wongseripipatana, Ph.D.)
………………………………………….……. Thesis Advisor
(Associate Professor Nijsiri Ruangrungsi, Ph.D.)
……………………………………………….. Thesis Co-Advisor
(Professor Somsak Ruchirawat, Ph.D.)
……………………………………………….. Thesis Co-Advisor
(Associate Professor Nongluksna Sriubolmas, Ph.D.)
……………………………………………….. Member
(Associate Professor Ekarin Saifah, Ph.D.)
……………………………………………….. Member
(Police Captain Suchada Sukrong, Ph.D.)
……………………………………………….. Member
(Associate Professor Wandee Gritsanapan, Ph.D.)
ศิรวิ รรณ อธิคมกุลชัย: องคประกอบทางเคมีและฤทธิ์ทางชีวภาพของตนสิรินธรวัลลีและตน
เปลาแพะ. (CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF
BAUHINIA SIRINDHORNIAE AND CROTON HUTCHINSONIANUS ) อาจารยทปี่ รึกษา:
รศ. ดร. นิจศิริ เรืองรังษี, อาจารยที่ปรึกษารวม: ศ.ดร. สมศักดิ์ รุจิรวัฒน, รศ.ดร. นงลักษณ
ศรีอบุ ลมาศ, 284 หนา. ISBN : 974-17-5964-9.
การศึกษาทางพฤกษเคมีของลําตนและรากสิรนิ ธรวัลลี สามารถแยกสารที่เคยมีรายงานมาแลวไดทั้งหมด
17 ชนิด ไดแก cyanoglucoside 2 ชนิด (lithospermoside และ menisdaurin), flavan 1 ชนิด ((-)-epicatechin),
flavanone 2 ชนิด ((2S)-naringenin และ (2S)-eriodictyol), flavanonol 1 ชนิด ((+)-taxifolin), flavone 1 ชนิด
(luteolin), chalcone 1 ชนิด (isoliquiritigenin), chromone 1 ชนิด (5,7-dihydroxychromone), chromone glucoside 1
ชนิด (5-hydroxychromone 7-β-D-glucoside), lignan glycoside 2 ชนิด ((+)-isolariciresinol 3α-O-α-L-rhamnoside
และ (+)-lyoniresinol 3α-O-α-L-rhamnoside), triterpenoid 2 ชนิด (lupeol และ glutinol), steroid glucoside 1 ชนิด
(sitosteryl-3-O-β-D-glucoside) และ สารกลุม phenolic 2 ชนิด (3,4,5-trimethoxyphenolic-1-O-β-D-glucoside และ
protocatechuic acid) สําหรับการศึกษาทางพฤกษเคมีของกิ่งและใบเปลาแพะสามารถแยกสารได 6 ชนิดซึ่งเปนสาร
ใหม 2 ชนิด คือ 3′-(4′′-hydroxy-3′′,5′′-dimethoxyphenyl)-propyl benzoate และ 3′-(4′′-hydroxyphenyl)-propyl
benzoate นอกจากนี้ยังพบสารที่มรี ายงานมาแลวอีก 4 ชนิด ไดแก farnesyl acetone, poilaneic acid 4hydroxybenzaldehyde และ dihydroconiferylbenzoate การพิสจู นสตู รโครงสรางทางเคมีของสารที่แยกไดนี้ อาศัย
การวิเคราะหสเปคโตรสโคป รวมกับการเปรียบเทียบขอมูลของสารที่ทราบโครงสรางแลว นอกจากนี้ยังไดนําสารที่
แยกไดไปทดสอบฤทธิ์ทางชีวภาพ ไดแก ฤทธิ์ตานแบคทีเรีย, ฤทธิ์ตา นเชื้อรา, ฤทธิ์ความเปนพิษตอเซลล และฤทธิ์
จับอนุมลู อิสระ พบวา (+)-isolariciresinol 3α-O-α-L-rhamnoside และ (+)-lyoniresinol 3α-O-α-L-rhamnoside มี
ฤทธิ์ในการจับอนุมลู อิสระ (2S)-eriodictyol และ isoliquiritigenin มีฤทธิ์ในการยับยั้งเชื้อ Bacillus subtilis และ
Staphylococcus aureus ในขณะที่ (2S)-naringenin และ luteolin มีฤทธิ์ในการยับยั้งเชื้อ B. subtilis นอกจากนี้ 3′(4′′-hydroxy-3′′,5′′-dimethoxyphenyl)-propyl, dihydroconiferyl benzoate และ 3′-(4′′-hydroxyphenyl)-propyl
benzoate แสดงฤทธิ์ระดับปานกลางในการยับยั้งเชื้อรา Candida albicans สวนการตรวจสอบฤทธิ์ความเปนพิษตอ
เซลลนั้นพบวา 3′-(4′′-hydroxy-3′′,5′′-dimethoxyphenyl)-propyl มีฤทธิ์ความเปนพิษตอเซลลมะเร็ง NCI-H187 ใน
ระดับต่ํา ในขณะที่ dihydroconiferylbenzoate และ 3′-(4′′-hydroxyphenyl)-propyl benzoate ไมมฤี ทธิ์ความเปนพิษ
ตอเซลลมะเร็ง NCI-H187
สาขาวิชา เภสัชเคมีและผลิตภัณฑธรรมชาติ
ปการศึกษา 2547
ลายมือชื่อนิสิต.........................................................
ลายมือชื่ออาจารยที่ปรึกษา......................................
ลายมือชื่ออาจารยที่ปรึกษารวม...............................
ลายมือชื่ออาจารยที่ปรึกษารวม...............................
# # 4276962633
: MAJOR: PHARMACEUTICAL CHEMISTRY AND NATURAL PRODUCTS
KEY WORD:
BAUHINIA SIRINDHORNIAE/ CROTON HUTCHINSONIANUS/ FLAVONOID/
CYANOGLUCOSIDE/
PHENYLPROPYL
BENZOATE/
FREE
RADICAL
SCAVENGING
ACTIVITY/ ANTIBACTERIAL ACTIVITY/ ANTIFUNGAL ACTIVITY/ CYTOTOXICITY
SIRIVAN ATHIKOMKULCHAI: CHEMICAL CONSTITUENTS AND BIOLOGICAL
ACTIVITIES OF BAUHINIA SIRINDHORNIAE AND CROTON HUTCHINSONIANUS.
THESIS ADVISOR: ASSOC. PROF. NIJSIRI RUANGRUNGSI, Ph. D. THESIS COADVISOR: PROF. SOMSAK RUCHIRAWAT, ASSOC. PROF. NONGLUKSNA
SRIUBOLMAS, Ph. D., pp. 284 ISBN: 974-17-5964-9
Phytochemical study of the stems and roots of Bauhinia sirindhorniae K. & SS. Larsen led to the
isolation of seventeen known compounds, two cyanoglucosides (lithospermoside and menisdaurin), one
flavan ((-)-epicatechin), two flavanones ((2S)-naringenin and (2S)-eriodictyol), one flavanonol ((+)taxifolin), one flavone (luteolin), one chalcone (isoliquiritigenin), one chromone (5,7-dihydroxychromone),
one chromone glucoside (5-hydroxychromone 7-β-D-glucoside), two lignan glycosides ((+)-isolariciresinol
3α-O-α-L-rhamnoside and (+)-lyoniresinol 3α-O-α-L-rhamnoside), two triterpenoids (lupeol and glutinol),
one
steroid
glucoside
(sitosteryl-3-O-β-D-glucoside)
and
other
trimethoxyphenolic-1-O-β-D-glucoside and protocatechuic acid).
phenolic
compounds
(3,4,5-
Additionally, six compounds were
obtained from the phytochemical investigation of the branches and leaves of Croton hutchinsonianus
Hosseus. These included two new compounds 3′-(4′′-hydroxy-3′′,5′′-dimethoxyphenyl)-propyl benzoate and
3′-(4′′-hydroxyphenyl)-propyl benzoate and other four known compounds, namely farnesyl acetone,
poilaneic acid, 4-hydroxybenzaldehyde and dihydroconiferylbenzoate. The structure determination of all
isolates were accomplished by spectroscopic methods and compared with the previously reported data of
known compounds. The isolated compounds were also subjected to biological evaluations, for antibacterial,
antifungal, cytotoxic and free radical scavenging activities. (+)-Isolariciresinol 3α-O-α-L-rhamnoside and
(+)-lyoniresinol 3α-O-α-L-rhamnoside showed free radical scavenging activity.
(2S)-Eriodictyol and
isoliquiritigenin showed activity against Bacillus subtilis and Staphylococcus aureus whereas (2S)naringenin and luteolin exhibited activity against Bacillus subtilis. Furthermore, 3′-(4′′-hydroxy-3′′,5′′dimethoxyphenyl)-propyl benzoate, dihydroconiferyl benzoate and 3′-(4′′-hydroxyphenyl)-propyl benzoate
revealed moderate antifungal activity against Candida albicans.
In addition, 3′-(4′′-hydroxy-3′′,5′′-
dimethoxyphenyl)-propyl benzoate showed weak cytotoxic activity against NCI-H187 cell line while
dihydroconiferylbenzoate and 3′-(4′′-hydroxyphenyl)-propyl benzoate were inactive.
Field of study Pharmaceutical Chemistry
Student’s signature………………………..
and Natural Products
Advisor’s signature………………………..
Academic year 2004
Co-Advisor’s signature……………………
Co-Advisor’s signature……………………
ACKNOWLEDGEMENTS
The author would like to express her sincere and grateful thanks to the following
institutions and people who encouraged and supported her research fulfillment:
Associate Professor Dr. Nijsiri Ruangrungsi, Department of Pharmacognosy, Faculty of
Pharmaceutical Sciences, Chulalongkorn University, for his valuable advices, keen interests and
encouragements throughout this study.
Professor Dr. Somsak Ruchirawat, Chulabhorn Research Institute, for his excellent advice
and kind assistance.
Associate Professor Dr. Nongluksna Sriubolmas, Department of Microbiology, Faculty of
Pharmaceutical Sciences, Chulalongkorn University, for her kindness, polite suggestions on the
determination of antimicrobial activities.
The thesis committee for their constuctive suggestions and criticial review for her thesis.
The Thailand Research Fund, for a 1999 Royal Golden Jubilee Scholarship and the
Association of International Education, Japan, for a research grant.
Professor Dr. Kazuei Igarashi, Department of Clinical Biochemistry, Faculty of
Pharmaceutical Sciences, Chiba University, for his kindly help and suggestions.
Associate Professor Dr. Fumio Ikegami, Laboratory of Plant Chemistry, Faculty of
Pharmaceutical Sciences, Chiba University, for his kind assistance and useful suggestions.
Research Associate Dr. Toshikazu Sekine, Department of Pharmacognosy, Faculty of
Pharmaceutical Sciences, Chiba University, for his valuable guidance and advices.
Dr. Megumi Sumino, Laboratory of Plant Chemistry, Faculty of Pharmaceutical Sciences,
Chiba University, for her kindly help and encouragement.
Dr. Hunsa Prawat and Miss Vilailak Prachayavarakorn, Laboratory of Natural products,
Chulabhorn Research Institute, for their kind assistance and guidance.
Dr. Nopporn Thasana, Dr. Thumnoon Mutarapat, Dr. Poonsakdi Ploypradith and
Dr. Duanpen Lertpibulpanya, Laboratory of Medicinal Chemistry, Chulabhorn Research Institute,
for their warm suggestions.
Staffs of the Chemical Analytical Center, Chiba University and the staffs of Chulabhorn
Research Institute (CRI) for collaboration and excellent support. The Biodiversity Research and
Training Program (BRT), Thailand for cytotoxicity and antifungal tests.
All members of the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences,
Chulalongkorn University, Laboratory of Plant Chemistry, Faculty of Pharmaceutical Sciences,
Chiba University, Laboratory of Medicinal Chemistry and Laboratory of Natural Products,
Chulabhorn Research Institute for their friendship, assistance and understanding.
Finally, the most special thanks to the author’s family and friends for their love and
continuous support.
CONTENTS
Page
ABSTRACT (Thai)………………………………………………………………
iv
ABSTRACT (English)…………………………………………………………...
v
ACKNOWLEDGEMENTS……………………………………………………...
vi
CONTENTS……………………………………………………………………...
vii
LIST OF TABLES……………………………………………………………….
xiii
LIST OF FIGURES……………………………………………………………...
xv
LIST OF SCHEMES…………………………………………………………….
xxi
LIST OF ABBREVIATIONS AND SYMBOLS………………………………..
xxii
CHAPTER
I INTRODUCTION……………………………………………………………..
1
II HISTORICAL
1. Chemical Constituents of Bauhinia spp…………………………………...
13
2. Chemical Constituents of Croton spp……………………………………..
30
3. Literature Reviews of Croton hutchinsonianus…………………………...
57
4. Biosynthetic Relationship of Flavonoids in Bauhinia spp………………...
58
5. Biosynthetic Relationship of Diterpenoids in Croton spp………………...
59
6. Traditional Uses and Biological Activities of Bauhinia spp……….…..…
60
7. Traditional Uses and Biological Activities of Croton spp……….…….…
61
III EXPERIMENTAL
1. Sources of Plant Materials………………………………………………...
64
2. General Techniques……………………………………………………….. 64
2.1 Analytical Thin-Layer Chromatography……………………………....
64
2.2 Preparative Thin Layer Chromatography………………………….….. 65
2.3 Column Chromatography
2.3.1
Vacuum Liquid Column Chromatography……………………. 65
2.3.2
Flash Column Chromatography……………………………….
65
2.3.3
Gel Filtration Chromatography………………………………..
65
2.3.4
High Pressure Liquid Chromatography……………………….. 66
2.4 Spectroscopy
viii
CONTENTS (continued)
Page
2.4.1
Ultraviolet (UV) Absorption Spectra………………………….
66
2.4.2
Infrared (IR) Absorption Spectra……………………………...
66
2.4.3
Mass Spectra……………………………...…………………...
66
2.4.4
Proton and Carbon-13 Nuclear Magnetic Resonance
(1H- and 13C-NMR) Spectra…………………………………...
67
2.5 Physical Properties
2.5.1
Optical Rotations……………………………………………… 67
2.5.2
Circular Dichoism (CD) Spectra………………………………
67
2.5.3
Melting Points…………………………...…………………….
67
2.6 Solvents………………………………………………………………
67
2.7 Chemicals…………………………………………………………….
67
2.8 Microtiter Plate Reader………………………...…………………….
67
3. Extraction and Isolation
3.1 Extraction and Isolation of the Stems of Bauhinia sirindhorniae....…. 68
3.1.1
Extraction……………………………………………………...
68
3.1.2 Isolation…………………………………………………….….
68
3.1.2.1 Isolation of Compounds from Chloroform Extract….…...
68
3.1.2.1.1 Isolation of Compound BSC1…….…………….…...
68
3.1.2.1.2 Isolation of Compound BSC2…………………….....
68
3.1.2.2 Isolation of Compounds from Butanol Extract…………..
69
3.1.2.2.1 Isolation of Compound BSB1 ………………………
69
3.1.2.2.2 Isolation of Compound BSB2……………………….
69
3.1.2.2.3 Isolation of Compound BSB3 ………………………
70
3.1.2.2.4 Isolation of Compound BSB4……………………….
70
3.1.2.2.5 Isolation of Compound BSB5……………………….
70
3.1.2.2.6 Isolation of Compound BSB6……………………….
71
3.2 Extraction and Isolation of the Roots of Bauhinia sirindhorniae..…..
71
3.2.1
Extraction……………………………………………………...
71
3.2.2
Isolation ……………………………………………………….
71
ix
CONTENTS (continued)
Page
3.2.2.1 Isolation of Compounds from Chloroform Extract….….…
3.2.2.1.1 Isolation of Compound BRC1…….…………….…..
71
72
3.2.2.1.2 Isolation of Compound BRC2…….…………….…..... 72
3.2.2.2 Isolation of Compounds from Butanol Extract….….….…
72
3.2.2.2.1 Isolation of Compound BRB1…….………………….. 72
3.2.2.2.2 Isolation of Compound BRB2…….………………….. 73
3.2.2.2.3 Isolation of Compound BRB3…….………………….. 73
3.2.2.2.4 Isolation of Compound BRB4…….………………….. 73
3.2.2.2.5 Isolation of Compound BRB5…….………………….. 74
3.2.2.2.1 Isolation of Compound BRB6…….………………….. 74
3.2.2.2.1 Isolation of Compound BRB7…….………………….. 75
3.3 Extraction and Isolation of the Leaves of Croton hutchinsonianus….
75
3.3.1
Extraction…………………………………………………..…
75
3.3.2
Isolation……………………………………………………….
75
3.3.2.1 Isolation of Compounds from Ethyl Acetate Extract…...…
75
3.3.2.1.1 Isolation of Compound CBE1…….…………….…….
76
3.3.2.1.2 Isolation of Compound CBE2…………..…….………
76
3.3.2.1.3 Isolation of Compound CBE4…………..…….………
76
3.4 Extraction and Isolation of the Branches of Croton hutchinsonianus.
77
3.4.1
Extraction…………………………………………………..….
77
3.4.2
Isolation…………………………………………………..……
77
3.4.2.1 Isolation of Compounds from Ethyl Acetate Extract….….
77
3.4.2.1.1 Isolation of Compound CBE1…….………..…………
77
3.4.2.1.2 Isolation of Compound CBE2…….………..…………
77
3.4.2.1.3 Isolation of Compound CBE3…….…..………………
78
3.4.2.1.4 Isolation of Compound CBE4……...…………………
78
3.4.2.1.5 Isolation of Compound CBE5…...……………………
78
3.4.2.1.6 Isolation of Compound CBE6…...……………………
79
4. Physical and Spectra Data of Isolated Compounds
x
CONTENTS (continued)
Page
4.1 Compound BSC1……………………………...……………………..
91
4.2 Compound BSC2……………………………...……………………..
91
4.3 Compound BSB1……………………………………...……………..
4.4 Compound BSB2……………………………………...……………..
91
92
4.5 Compound BSB3……………………………………...……………..
92
4.6 Compound BSB4……………………………………...……………..
92
4.7 Compound BSB5……………………………………...……………..
93
4.8 Compound BSB6…………………………………………………….
93
4.9 Compound BRC1…………………………………………………….
93
4.10 Compound BRC2……………………………………………………
94
4.11 Compound BRB1………………………………………...………….
94
4.12 Compound BRB2………………………………………...………….
94
4.13 Compound BRB3………………………………………...………….
95
4.14 Compound BRB4………………………………………...………….
95
4.15 Compound BRB5………………………………………...………….
96
4.16 Compound BRB6………………………………………...………….
96
4.17 Compound BRB7………………………………………...………….
96
4.18 Compound CBE1………………………………………...………….
97
4.19 Compound CBE2………………………………………...………….
97
4.20 Compound CBE3………………………………………...………….
97
4.21 Compound CBE4………………………………………...………….
98
4.22 Compound CBE5………………………………………...………….
98
4.23 Compound CBE6………………………………………...………….
99
5. Evaluation of Biological Activities……………………………...………….
99
5.1 Antimicrobial Activity…………………………………...…………..
99
5.1.1
Agar Diffusion Assay…………………………..……………. 99
5.1.1.1 Preparation of Sample……………………..……………..
99
5.1.1.2 Preparation of the Inoculum……………..………………. 99
5.1.1.3 Preparation of Test Plates……………………...……….... 100
xi
CONTENTS (continued)
Page
5.1.1.3.1 Preparation for Testing Bacteria…………….……… 100
5.1.1.3.2 Preparation for Testing Fungi…………….………… 100
5.1.1.4 Inoculation of Agar Plates…………………..………….... 100
5.1.1.5 Assay Procedure…………………………..……………... 100
5.1.2
Determination of MIC and MBC…………………………..... 101
5.1.2.1 Test Samples Dilution……………………………….…... 101
5.1.2.2 Preparation of the Inoculum………………………...…… 101
5.1.2.3 Assay Procedure…………………………………...…….. 101
5.2 Determination of Free Radical Scavenging Activity…………...……. 101
5.2.1
TLC Screening Assay………………………………………… 101
5.2.2 Free Radical Scavenging Activity Assay……………………... 101
5.2.2.1 Preparation of Test Sample………………………………... 102
5.2.2.2 Preparation of DPPH Solution………………………….…. 102
5.2.2.3 Measurement of Activity……………………………....….. 103
5.3 Cytotoxic Activity…………………………………………….……… 103
5.4 Antifungal Activity…………………………………………...……… 103
IV RESULTS AND DISCUSSION
1. Structure Determination of Isolated Compounds
1.1 Structure Determination of Compound BSC1………………………... 105
1.2 Structure Determination of Compound BSC2………………………... 108
1.3 Structure Determination of Compound BSB1………………………... 110
1.4 Structure Determination of Compound BSB2………………………... 112
1.5 Structure Determination of Compound BSB3………………………... 114
1.6 Structure Determination of Compound BSB4………………………... 116
1.7 Structure Determination of Compound BSB5………………………... 119
1.8 Structure Determination of Compound BSB6………………………... 121
1.9 Structure Determination of Compound BRC1………………………... 124
1.10 Structure Determination of Compound BRC2……………………..... 126
1.11 Structure Determination of Compound BRB1……………………..... 128
xii
CONTENTS (continued)
Page
1.12 Structure Determination of Compound BRB2……………………..... 131
1.13 Structure Determination of Compound BRB3……………………..... 133
1.14 Structure Determination of Compound BRB4……………………....
135
1.15 Structure Determination of Compound BRB5…………………..…..
137
1.16 Structure Determination of Compound BRB6………………...…….. 139
1.17 Structure Determination of Compound BRB7………………………. 141
1.18 Structure Determination of Compound CBE1……………………..... 143
1.19 Structure Determination of Compound CBE2……………………..... 145
1.20 Structure Determination of Compound CBE3……………………..... 149
1.21 Structure Determination of Compound CBE4……………………..... 151
1.22 Structure Determination of Compound CBE5……………………..... 154
1.23 Structure Determination of Compound CBE6……………………..... 157
2. Biological Activities of Compounds from Bauhinia sirindhorniae
2.1 Antimicrobial Activity……………………………………………..…. 160
2.2 Free Radical Scavenging Activity………………………………..…… 161
3. Biological Activities of Compounds from Croton hutchinsonianus
3.1 Cytotoxic Activity………………………………………………..…… 162
3.2 Antifungal Activity…………………………………………….……... 163
V CONCLUSION………………………………………………………...……... 164
REFERENCES…………………………………………………………..….……. 168
APPENDICES……………………………………………………………..…….. 179
VITA…………………………………………………………………………..…
259
xiii
LIST OF TABLES
Table
Page
1
Distribution of flavonoids in Bauhinia spp…………..………….………..
13
2
Distribution of steroids in Bauhinia spp………………………..………...
20
3
Distribution of miscellaneous compounds in Bauhinia spp.……………..
24
4
Distribution of diterpenes in Croton spp……………….…….…………..
30
5
Distribution of triterpenes in Croton spp……………………….………..
51
6
Distribution of alkaloids in Croton spp….………………….……………
52
7
Distribution of miscellaneous compounds in Croton spp………………..
54
8
NMR Spectral data of compound BSC1 and lupeol (in CDCl3)…………. 107
9
NMR Spectral data of compound BSC2 and glutinol (in CDCl3)……….. 109
10
NMR Spectral data of compound BSB1
and isoliquiritigenin (in DMSO-d6)……………………………………...
11
111
NMR Spectral data of compound BSB2
and (+)-isolariciresinol-3α-O-α-L- rhamnoside (in CD3OD)……………. 113
12
NMR Spectral data of compound BSB3
and 3,4,5-trimethoxyphenolic-1-O-β-D-glucoside (in CD3OD)…………. 115
13
NMR Spectral data of compound BSB4 (in CD3OD)
and (-)-epicatechin (in DMSO-d6)……………………………………….. 118
14
NMR Spectral data of compound BSB5 (in CD3OD)
and protocatechuic acid (in acetone-d6)……………….…………………. 120
15
NMR Spectral data of compound BSB6
and lithospermoside (in D2O)……………… ……………………………. 123
16
NMR Spectral data of compound BRC1
and 5,7-dihydroxychromone (in CD3OD)………………………………... 125
17
NMR Spectral data of compound BRC2 (in CDCl3 + CD3OD)
and sitosteryl-3-O-β-D-glucoside (in pyridine-d5)………….…………… 127
18
19
NMR Spectral data of compound BRB1 (in CD3OD)
and (2S)-naringenin (in acetone-d6)……………………………………..
130
NMR Spectral data of compound BRB2 and luteolin (in DMSO-d6)…..
132
xiv
LIST OF TABLES (continued)
Table
20
Page
NMR Spectral data of compound BRB3 (in CD3OD)
and (2S)-eriodictyol (in DMSO-d6)……………………………………… 134
21
NMR Spectral data of compound BRB4 and (+)-taxifolin (in CD3OD)… 136
22
NMR Spectral data of compound BRB5 (in CD3OD)
and (+)-lyoniresinol-3α-O-α-L-rhamnoside (in pyridine-d5)……………
23
138
NMR Spectral data of compound BRB6
and 5-hydroxychromone-7-β-D-glucoside (in CD3OD)…………………
140
24
NMR Spectral data of compound BRB7 and menisdaurin (in CD3OD)...
142
25
NMR Spectral data of compound CBE1 and farnesyl acetone (in CDCl3). 144
26
NMR Spectral data of compound CBE2 and poilaneic acid (in CDCl3)… 148
27
NMR Spectral data of compound CBE3 (in CDCl3)…………………….. 150
28
NMR Spectral data of compound CBE4 (in CDCl3)…………………….. 153
29
NMR Spectral data of compound CBE5 (in CDCl3)…………………….. 156
30
NMR Spectral data of compound CBE6 (in CDCl3)…………………….. 159
31
Antibacterial activity of some isolated compounds from
Bauhinia sirindhorniae…………………………………………………... 161
32
The DPPH radical scavenging activity of compounds [222] and [215]…. 162
33
The cytotoxic activity of the crude extracts of C. hutchinsonianus……… 162
34
Compounds isolated from chloroform extract of the stems of
Bauhinia sirindhorniae…………………………………………………… 165
35
Compounds isolated from butanol extract of the stems of
Bauhinia sirindhorniae…………………………………………………… 165
36
Compounds isolated from chloroform extract of the roots of
Bauhinia sirindhorniae……………………………………………………. 166
37
Compounds isolated from butanol extract of the roots of
Bauhinia sirindhorniae……………………………………………………. 166
38
Compounds isolated from ethyl acetate extract of
Croton hutchinsonianus………………………………………………….. 167
xv
LIST OF FIGURES
Figure
Page
1
Bauhinia sirindhorniae K. & S.S. Larsen………………………………...
11
2
Croton hutchinsonianus Hosseus………………………………………...
12
3
Structures of compounds isolated from the stems of
Bauhinia sirindhorniae………………………………………………..….. 88
4
Structures of compounds isolated from the roots of
Bauhinia sirindhorniae…………………………………………………...
5
89
Structures of compounds isolated from the leaves and branches of
Croton hutchinsonianus………………………………………………......
90
6
NOESY experiment in compound CBE2……………………………..…. 147
7
1
H-1H COSY and HMBC correlations of compound CBE4………..…....
152
8
1
H-1H COSY and HMBC correlations of compound CBE5…….…….…
155
9
1
H-1H COSY and HMBC correlations of compound CBE6….……….…
158
10
IR Spectrum of compound BSC1 (KBr disc)………………………….… 180
11
EIMS Mass spectrum of compound BSC1………………………….…… 180
12
1
13
13
14
IR Spectrum of compound BSC2 (KBr disc)……………………….…… 182
15
EIMS Mass spectrum of compound BSC2………………………………
16
1
17
13
18
UV Spectrum of compound BSB1 (MeOH)…………………………..…. 184
19
IR Spectrum of compound BSB1 (KBr disc)………………………..…… 184
20
FAB+MS Mass spectrum of compound BSB1…………………………… 185
21
1
22
13
23
1
24
HMQC Spectrum of compound BSB1 (DMSO-d6)……………………… 187
25
HMBC Spectrum of compound BSB1 (DMSO-d6)……………………… 187
26
UV Spectrum of compound BSB2 (MeOH)…………………………...… 188
27
IR Spectrum of compound BSB2 (KBr disc)………………………..…… 188
H NMR (500 MHz) Spectrum of compound BSC1 (CDCl3)………..….
C NMR (125 MHz) Spectrum of compound BSC1 (CDCl3)………..…
181
181
182
H NMR (500 MHz) Spectrum of compound BSC2 (CDCl3)…………… 183
C NMR (125 MHz) Spectrum of compound BSC2 (CDCl3)………...… 183
H NMR (400 MHz) Spectrum of compound BSB1 (DMSO-d6)……..…. 185
C NMR (100 MHz) Spectrum of compound BSB1 (DMSO-d6)….…… 186
H-1H COSY Spectrum of compound BSB1 (DMSO-d6)………….……. 186
xvi
LIST OF FIGURES (continued)
Figure
Page
28
FAB+MS Mass spectrum of compound BSB2…………………………… 189
29
1
30
13
31
UV Spectrum of compound BSB3 (MeOH) )……………………………. 190
32
IR Spectrum of compound BSB3 (KBr disc)…………………………….. 191
33
FAB+MS Mass spectrum of compound BSB3…………………………… 191
34
1
35
13
36
HMQC Spectrum of compound BSB3 (CD3OD)………………………... 193
37
HMBC Spectrum of compound BSB3 (CD3OD)………………………... 193
38
UV Spectrum of compound BSB4 (MeOH)……………………………... 194
39
IR Spectrum of compound BSB4 (KBr disc)……………………………. 194
40
FAB-MS Mass spectrum of compound BSB4…………………………… 195
41
1
42
13
43
1
44
HMQC Spectrum of compound BSB4 (CD3OD)………………………... 197
45
HMBC Spectrum of compound BSB4 (CD3OD)………………….…….. 197
46
UV Spectrum of compound BSB5 (MeOH)…………………………..…
47
IR Spectrum of compound BSB5 (KBr disc)………………………..…... 198
48
FAB+MS Mass spectrum of compound BSB5……………………..…….
49
1
50
13
51
UV Spectrum of compound BSB6 (water)………………….…………… 200
52
IR Spectrum of compound BSB6 (KBr disc)………………..…………… 201
53
FAB+MS Mass spectrum of compound BSB6…………………………… 201
54
1
55
13
56
HMQC Spectrum of compound BSB6 (D2O)……………………….…… 203
H-NMR (500 MHz) Spectrum of compound BSB2 (CD3OD)………….. 189
C-NMR (125 MHz) Spectrum of compound BSB2 (CD3OD)…………. 190
H NMR (500 MHz) Spectrum of compound BSB3 (CD3OD)………….. 192
C NMR (125 MHz) Spectrum of compound BSB3 (CD3OD)….……… 192
H NMR (500 MHz) Spectrum of compound BSB4 (CD3OD)………….. 195
C NMR (125 MHz) Spectrum of compound BSB4 (CD3OD)….……… 196
H-1H COSY Spectrum of compound BSB4 (CD3OD)………….………. 196
198
199
H NMR (500 MHz) Spectrum of compound BSB5 (CD3OD)…..……… 199
C NMR (125 MHz) Spectrum of compound BSB5 (CD3OD)..………..
200
H NMR (500 MHz) Spectrum of compound BSB6 (D2O)…………….... 202
C NMR (125 MHz) Spectrum of compound BSB6 (D2O)….………..… 202
xvii
LIST OF FIGURES (continued)
Figure
Page
57
HMBC Spectrum of compound BSB6 (D2O)…………………….……… 203
58
UV Spectrum of compound BRC1 (MeOH)……………………………... 204
59
IR Spectrum of compound BRC1 (KBr disc)……………………………. 204
60
FAB+MS Mass spectrum of compound BRC1…………………………... 205
61
1
H NMR (500 MHz) Spectrum of compound BRC1 (CD3OD)………….. 205
62
1
H NMR (500 MHz) Spectrum of compound BRC1 (acetone-d6)……..... 206
63
13
64
HMQC Spectrum of compound BRC1 (acetone-d6)………………..…… 207
65
HMBC Spectrum of compound BRC1 (acetone-d6)……………...……… 207
66
IR Spectrum of compound BRC2 (KBr disc)…………………….……… 208
67
FAB+MS Mass spectrum of compound BRC2………………….……….. 208
68
1
69
13
70
UV Spectrum of compound BRB1 (MeOH)…………………………...… 210
71
IR Spectrum of compound BRB1 (KBr disc)……………………………. 210
72
FAB+MS Mass spectrum of compound BRB1…………………………... 211
73
1
74
13
75
HMQC Spectrum of compound BRB1 (CD3OD)……………………...… 212
76
HMBC Spectrum of compound BRB1 (CD3OD)………………………... 213
77
UV Spectrum of compound BRB2 (MeOH)…………………………….. 213
78
IR Spectrum of compound BRB2 (KBr disc)……………………………. 214
79
FAB+MS Mass spectrum of compound BRB2…………………………... 214
80
1
81
13
82
HMQC Spectrum of compound BRB2 (DMSO-d6)……………………... 216
83
HMBC Spectrum of compound BRB2 (DMSO-d6)…………………….... 216
84
UV Spectrum of compound BRB3 (MeOH)……………………………... 217
85
IR Spectrum of compound BRB3 (KBr disc)……………………………. 217
C NMR (125 MHz) Spectrum of compound BRC1 (CD3OD)….…..…. 206
H NMR (500 MHz) Spectrum of compound BRC2 (CDCl3+CD3OD)…. 209
C NMR (125 MHz) Spectrum of compound BRC2 (CDCl3+CD3OD)… 209
H NMR (500 MHz) Spectrum of compound BRB1 (CD3OD)………….. 211
C NMR (125 MHz) Spectrum of compound BRB1 (CD3OD)….…….... 212
H NMR (500 MHz) Spectrum of compound BRB2 (DMSO-d6)……….. 215
C NMR (125 MHz) Spectrum of compound BRB2 (DMSO-d6)….…… 215
xviii
LIST OF FIGURES (continued)
Figure
Page
86
FAB+MS Mass spectrum of compound BRB3…………………………... 218
87
1
88
13
89
HMQC Spectrum of compound BRB3 (CD3OD)………………………..
90
HMBC Spectrum of compound BRB3 (CD3OD)………………………... 220
91
UV Spectrum of compound BRB4 (MeOH)…………………………….. 220
92
IR Spectrum of compound BRB4 (KBr disc)……………………………. 221
93
FAB+MS Mass spectrum of compound BRB4…………………………... 221
94
1
95
13
96
HMQC Spectrum of compound BRB4 (CD3OD)………………………... 223
97
HMBC Spectrum of compound BRB4 (CD3OD)………………………... 223
98
UV Spectrum of compound BRB5 (MeOH)…………………………….. 224
99
IR Spectrum of compound BRB5 (KBr disc)……………………………. 224
H NMR (500 MHz) Spectrum of compound BRB3 (CD3OD)……….…. 218
C NMR (125 MHz) Spectrum of compound BRB3 (CD3OD)….……... 219
219
H NMR (500 MHz) Spectrum of compound BRB4 (CD3OD)………….. 222
C NMR (125 MHz) Spectrum of compound BRB4 (CD3OD)….……… 222
100 FAB+MS Mass spectrum of compound BRB5…………………………... 225
101
1
102
13
103
1
H NMR (500 MHz) Spectrum of compound BRB5 (CD3OD)…………. 225
C NMR (125 MHz) Spectrum of compound BRB5 (CD3OD)….……... 226
H-1H COSY Spectrum of compound BRB5 (CD3OD)…………………. 226
104 HMQC Spectrum of compound BRB5 (CD3OD)………………………... 227
105 HMBC Spectrum of compound BRB5 (CD3OD)………………………... 227
106 UV Spectrum of compound BRB6 (MeOH)……………………………... 228
107 FAB+MS Mass spectrum of compound BRB6…………………………... 228
108
1
109
13
H NMR (500 MHz) Spectrum of compound BRB6 (CD3OD)………….. 229
C NMR (125 MHz) Spectrum of compound BRB6 (CD3OD)….…..…. 229
110 UV Spectrum of compound BRB7 (MeOH)………………………..…… 230
111 IR Spectrum of compound BRB7 (KBr disc)……………………..……... 230
112 FAB+MS Mass spectrum of compound BRB7…………………..………. 231
113
1
114
13
H NMR (500 MHz) Spectrum of compound BRB7 (CD3OD)..………… 231
C NMR (125 MHz) Spectrum of compound BRB7 (CD3OD)...……….. 232
xix
LIST OF FIGURES (continued)
Figure
115
1
Page
H-1H COSY Spectrum of compound BRB7 (CD3OD)…………………. 232
116 HMQC Spectrum of compound BRB7 (CD3OD)……………….……….. 233
117 HMBC Spectrum of compound BRB7 (CD3OD)…………….………….. 233
118 IR Spectrum of compound CBE1 (neat)…………………………………. 234
119 EIMS Mass spectrum of compound CBE1…………………….………… 234
120
1
121
13
122
1
H NMR (400 MHz) Spectrum of compound CBE1 (CDCl3)…………… 235
C NMR (100 MHz) Spectrum of compound CBE1 (CDCl3)...………… 235
H-1H COSY Spectrum of compound CBE1 (CDCl3)…………………… 236
123 HMQC Spectrum of compound CBE1 (CDCl3)…………………………. 236
124 HMBC Spectrum of compound CBE1 (CDCl3)…………………………. 237
125 UV Spectrum of compound CBE2 (MeOH)……………………………..
237
126 IR Spectrum of compound CBE2 (neat)…………………………………
238
127 EIMS Mass spectrum of compound CBE2………………………………. 238
128
1
129
13
130
1
H NMR (500 MHz) Spectrum of compound CBE2 (CDCl3)…………… 239
C NMR (125 MHz) Spectrum of compound CBE2 (CDCl3)….……….
239
H-1H COSY Spectrum of compound CBE2 (CDCl3)…………………... 240
131 NOESY Spectrum of compound CBE2 (CDCl3)………………………...
240
132 HMQC Spectrum of compound CBE2 (CDCl3)…………………………. 241
133 HMBC Spectrum of compound CBE2 (CDCl3)…………………………. 241
134 UV Spectrum of compound CBE3 (MeOH)……………………………..
242
135 IR Spectrum of compound CBE3 (neat)…………………………………
242
136 EIMS Mass spectrum of compound CBE3………………………………. 243
137
1
138
13
139
1
H NMR (400 MHz) Spectrum of compound CBE3 (CDCl3)…………… 243
C NMR (100 MHz) Spectrum of compound CBE3 (CDCl3)….……….. 244
H-1H COSY Spectrum of compound CBE3 (CDCl3)…………………… 244
140 HMQC Spectrum of compound CBE3 (CDCl3)……………………...….. 245
141 HMBC Spectrum of compound CBE3 (CDCl3)……………………...….. 245
142 UV Spectrum of compound CBE4 (MeOH)……………………………..
246
143 IR Spectrum of compound CBE4 (neat)…………………………………. 246
144 EIMS Mass spectrum of compound CBE4………………………………. 247
xx
LIST OF FIGURES (continued)
Figure
Page
145
1
H NMR (400 MHz) Spectrum of compound CBE4 (CDCl3)…………… 247
146
1
H NMR (400 MHz) Spectrum of compound CBE4 (CDCl3 + D2O)……. 248
147
13
148
1
C NMR (100 MHz) Spectrum of compound CBE4 (CDCl3)….……….. 248
H-1H COSY Spectrum of compound CBE4 (CDCl3)…………………… 249
149 HMQC Spectrum of compound CBE4 (CDCl3)………………………..... 249
150 HMBC Spectrum of compound CBE4 (CDCl3)…………………………. 250
151 UV Spectrum of compound CBE5 (MeOH)……………………………... 250
152 IR Spectrum of compound CBE5 (neat)…………………………………. 251
153 EIMS Mass spectrum of compound CBE5…………………………..…..
154
1
155
13
156
1
H NMR (400 MHz) Spectrum of compound CBE5 (CDCl3)……..…….
C NMR (100 MHz) Spectrum of compound CBE5 (CDCl3)…...……...
251
252
252
1
H- H COSY Spectrum of compound CBE5 (CDCl3)…………..………. 253
157 HMQC Spectrum of compound CBE5 (CDCl3)……………….………...
253
158 HMBC Spectrum of compound CBE5 (CDCl3)…………….…………… 254
159 UV Spectrum of compound CBE6 (MeOH)……………….…………….
254
160 IR Spectrum of compound CBE6 (neat)………………….……………… 255
161 EIMS Mass spectrum of compound CBE6…………….………………… 255
162
1
163
13
164
1
H NMR (400 MHz) Spectrum of compound CBE6 (CDCl3)…………… 256
C NMR (100 MHz) Spectrum of compound CBE6 (CDCl3)….……….
256
H-1H COSY Spectrum of compound CBE6 (CDCl3)…………………… 257
165 HMQC Spectrum of compound CBE6 (CDCl3)…………………….…… 257
166 HMBC Spectrum of compound CBE6 (CDCl3)…………………….…… 258
xxi
LIST OF SCHEMES
Scheme
Page
1
Currently proposed interrelationships between flavonoid monomer…….
58
2
Biosynthetic relationship of diterpenes in Croton spp…………………...
59
3
Separation of the CHCl3 extract of the stems of
Bauhinia sirindhorniae…………………………………………………...
4
Separation of the butanol extract of the stems of
Bauhinia sirindhorniae…………………………………………………...
5
85
Separation of the ethyl acetate extract of the leaves of
Croton hutchinsonianus…………………………………………………..
10
84
Separation of fraction RB-D from the butanol extract of
the roots of Bauhinia sirindhorniae…………………………..………….
9
83
Separation of the butanol extract of the roots of
Bauhinia sirindhorniae……………………………………………………
8
82
Separation of the CHCl3 extract of the roots of
Bauhinia sirindhorniae…………………………………………………...
7
81
Separation of fraction SB-C from the butanol extract of
the stems of Bauhinia sirindhorniae…………………………..………….
6
80
86
Separation of the ethyl acetate extract of the branches of
Croton hutchinsonianus…………………………………………………...
87
11
EIMS Spectra fragmentations of compound BSC1……………………… 106
12
EIMS Spectra fragmentations of compound CBE4……………………… 152
13
EIMS Spectra fragmentations of compound CBE5…………………….... 155
14
EIMS Spectra fragmentations of compound CBE6…………………….... 158
xxii
LIST OF ABBREVIATIONS AND SYMBOLS
[α] D23
=
Specific rotation at 23 °C and sodium D line (589 nm)
α
=
Alpha
acetone-d6
=
Deuterated acetone
β
=
Beta
br
=
Broad
°C
=
Degree Celsius
calcd
=
Calculated
CD
=
Circular Dichroism
CDCl3
=
Deuterated chloroform
CD3OD
=
Deuterated methanol
CHCl3
=
Chloroform
CH3CN
=
Acetronitrile
cm
=
Centimeter
cm-1
=
Reciprocal centimeter (unit of wave number)
13
=
Carbon-13 Nuclear Magnetic Resonance
d
=
Doublet (for NMR spectra)
1D
=
One Dimentional
2D
=
Two Dimentional
dd
=
Doublet of doublets (for NMR spectra)
ddd
=
Doublet of doublet of doublet (for NMR spectra)
dddd
=
Doublet of doublet of doublet of doublet (for NMR spectra)
dq
=
Doublet of quartet (for NMR spectra)
DEPT
=
Distortionless Enhancement by Polarization Transfer
D2O
=
Deuterated Water
DPPH
=
1,1-Diphenyl-2-picrylhydrazyl
δ
=
Chemical shift
ED50
=
50% Effective Dose
EIMS
=
Electron Impact Mass Spectrometry
EtOAc
=
Ethyl acetate
EtOH
=
Ethanol
C NMR
xxiii
LIST OF ABBREVIATIONS AND SYMBOLS (continued)
FABMS
=
Fast Atom Bombardment Mass Spectrometry
+
FAB MS
=
Fast Atom Bombardment Mass Spectrometry (positive mode)
-
FAB MS
=
Fast Atom Bombardment Mass Spectrometry (negative mode)
Fr.
=
Fraction
g
=
Gram
GGPP
=
Geranylgeranyl pyrophosphate
hr
=
Hour
1
H NMR
=
Proton Nuclear Magnetic Resonance
1
H-1H-COSY =
Homonuclear (Proton-Proton) Correlation Spectroscopy
HMBC
=
1
H-detected Heteronuclear Multiple Bond Coherence
HMQC
=
1
H-detected Heteronuclear Multiple Quantum Coherence
H2O
=
Water
HPLC
=
High Performance Liquid Chromatography
HRFABMS
=
High Resolution Fast Atom Bombardment Mass Spectrometry
Hz
=
Hertz
IC50
=
Median Inhibitory Concentration
IR
=
Infrared Spectrum
J
=
Coupling constant
KBr
=
Potassium bromide
KB
=
Human oral epidermoid carcinoma cell line
Kg
=
Kilogram
L
=
Liter
λmax
=
Wavelength at maximal absorption
ε
=
Molar absorptivity
m
=
Multiplet (for NMR spectra)
µg
=
Microgram
µL
=
Microliter
µM
=
Micromolar
MeOH
=
Methanol
mg
=
Milligram
xxiv
LIST OF ABBREVIATIONS AND SYMBOLS (continued)
[M+H]+
=
Protonated molecule
[M-H]-
=
Deprotonated molecule
=
Sodium adduct ion
[M+K]
=
Potassium adduct ion
MBC
=
Minimum Bactericidal Concentration
MHz
=
Megahertz
MIC
=
Minimum Inhibition Concentration
min
=
Minute
mL
=
Milliliter
mM
=
Millimolar
m.p.
=
Melting point
MW
=
Molecular weight
m/z
=
Mass to charge ratio
MS
=
Mass Spectrometry
MTT
=
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
mult.
=
Multiplicity
NCI-H187
=
Human small cell lung cancer cell line
nm
=
Nanometer
NMR
=
Nuclear Magnetic Resonance
NOESY
=
Nuclear Overhauser Effect Spectroscopy
o
=
Ortho
p
=
Para
P-388
=
Murine leukemia cell line
ppm
=
Part per million
PTLC
=
Preparative Thin Layer Chromatography
pyridine-d5
=
Deuterated pyridine
νmax
=
Wave number at maximal absorption
s
=
Singlet (for NMR spectra)
spp.
=
Species
t
=
Triplet (for NMR spectra)
[M+Na]+
+
xxv
LIST OF ABBREVIATIONS AND SYMBOLS (continued)
t-DCTN
=
Trans-dehydrocrotonin
TEAC
=
Trolox Equivalent Antioxidant Activity
TLC
=
Thin Layer Chromatography
Trolox
=
6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
UV
=
Ultraviolet
UV-VIS
=
Ultraviolet and Visible Spectrophotometry
V-79
=
A Chinese hamster lung cell line
CHAPTER I
INTRODUCTION
The genus Bauhinia belongs to the family Leguminosae of the subfamily
Caesalpinoidae. This genus consists of about 300 species distributed in Africa, Asia,
and Latin America.
Plants in the genus Bauhinia are trees, shrubs and tendrilled climbers. Leaves
are simple, entire, emarginated, bilobed or divided in two free leaflets. The midrib
often bristles between the lobes and the base often contains two darker alveoles.
Stipules are normally early carducous. Flowers are bisexual (rarely unisexual) with a
more or less pronounced receptacle. Calyx is five-merous, cup-shaped, spathaceous
or splitting into free segments during anthesis.
There are typically five petals.
Stamens are 10, 5, 3, 2 or 1 and anthers are released via longitudinal slits in all Thai
species except B. bidentata where they are released via a central pore. Ovary is
stipitate and is rarely sessile. Pods are dehiscent and are rarely indehiscent (Larsen,
Larsen and Vidal, 1984).
According to Smitinand (2001), the species of genus Bauhinia found in
Thailand are as follows:
B. acuminata L.
กาแจะกูโด Ka-chae-ku-do (Malay-Narathiwat);
กาหลง Kalong (Central);โยธิกา Yo thika (Nakhon
Si Thammarat); สมเสี้ยว Som siao (Central);เสี้ยว
นอย Siao noi (Chaing Mai).
B. aureifolia K. & S.S. Larsen
ใบสีทอง Bai si thong, ยานดาโอะ
Yan da o
(Narathiwat).
B. bassacensis Pierre ex Gagnep.
เครือเขาหนัง Khruea khao nang (Lampang);
ชงโค Chong kho, โยธิกา Yo thi ka (Peninsular); เถา
กระไดลิง Thao kradai ling (Southeastern).
B. bidentata Jack
ชงโคปาดอกแดง Chong kho pa dok daeng
subp. bicornuta (Miq.)
(Peninsular); เล็บกระรอก Lep krarok (Pattani)
2
K. & S.S. Larsen
เล็บควายเหล็ก Lep khwai lek (Yala).
B. binata Blanco
แสลงพัน Salaeng phan (Chon Buri).
B. bracteata (Graham ex Benth.)
ปอแกว Po-kaeo (Karen-Northern); ปอเจี๋ยน Po
Baker
chian (Northern); ปอบุง Po bung (Chaing Mai);
เสี้ยวเครือ Siao khruea (Nakhon Ratchasima); เสี้ยว
ดอกขาว Siao dok khao; เสี้ยวเตี้ย Siao tia (Loei);
เสี้ยวสม Siao som (Uthai Thani, Sakon nakhon);
แสลงพัน Saleang phan (Chon buri).
chrysophylla K. & S.S. Larsen
= B. aureifolia K. & S.S. Larsen
B. curtisii Prain
เครือเขาแกบ Khruea khao kaep (Northeastern).
decipiens Craib
= B. pottsii G.Don var. decipiens (Craib) K. &
S.S. Larsen
detergens Craib
= B. bassacensis Pierre ex Gagnep.
elongata Korth.
= B. pottsii G. Don var. pottsii
ยานตีนควาย Yan tin khwai (Narathiwat).
B. ferruginea Roxb.
flammifera Ridl.
= B. integrifolia Roxb.
B. glauca (Wall. Ex Benth.)
ชงโค Chong kho (Penninsular).
subsp. tenuiflora
คางโค Khang kho (Chanthaburi); พาซิว Pha-sio
(Watt ex C.B. Clarke)
(Karen-Lampang); เสี้ยวเครือ Siao khruea (Chiang
K. & S.S. Larsen
Mai, Lampang); เสี้ยวตน Siao ton (Nan); เสี้ยวปา
Siao pa (Chiang Mai)
ชงโคขี้ไก Chong kho khi kai (Kanchanaburi); เสี้ยว
B. harmsiana Hosseus
Siao (Phrae); เสี้ยวเคือ Siao khuea (Lamphun).
helferi Craib
B. hirsuta Weinm.
=
B. bracteata (Graham ex Benth.) Baker
วุงพู Wung-Phu (Karen-Mae Hong Son); เสี้ยวนอย
Siao noi (Northern).
3
horsfieldii (Miq.) MacBr.
= B. scandens L. var. horsfieldii (Miq.) K. & S.S.
Larsen
กุกูกูดอ Ku-ku-ku-do, กุกูกูบา Ku-ku-ku-ba (Malay-
B. integrifolia Roxb.
Pattani); ชงโคยาน Chong kho yan, ยานชงโค Yan
chong kho (Trang); ชิงโคยาน Ching kho yan
(Peninsular); ดาโอะ Da o (Narathiwat); เถาไฟ Thao
fai, โยทะกา Yo thaka (Bangkok); ปอลิง Po ling
(Surat thani); เล็บควายใหญ Lep khwai yai (Yala,
Pattani).
แสลงพัน Saleang phan (Kanchanaburi, Saraburi).
B. involucellata Kurz
kerrii Gagnep.
=
B. ornata Kurz var. kerii (Gagnep.) K. & S.S.
Larsen
B. lakhonensis Gagnep.
สมเสี้ยวเถา Som siao thao (Northeastern).
B. malabarica Roxb.
คังโค Khang kho (Suphan Buri); แดงโค Daeng kho
(Saraburi); ปาม Pam (Suai-Surin); สมเสี้ยว Som
siao (Northern); เสี้ยวสม Siao som (Nakhon
Ratchasima); เสี้ยวใหญ Siao yai (Prachin Buri).
media Craib
B. monandra Kurz.
= B. harmsiana Hosseus
จงโค Chong kho, โยทะกา Yo thaka (Bangkok)
One stamened bauhinia.
B. nervosa (Wall. Ex Benth.)
เสี้ยวแกว Siao kaeo (General).
Baker
B. ornata Kurz var. kerrii
กวาวขน Kwao khon (Chaing Mai); โคคลาน Kho
(Gagnep.) K. & S.S. Larsen
khlan (Prachuap Khiri Khan);ปอมุง Po mung
(Chiang Mai); เสี้ยว Siao; ชงโค Chong kho
(Phrae); เสี้ยวเครือ Siao khruea (Sukothai); แสลงพัน
แดง Saleang phan daeng (Loei, Lop Buri).
4
var. burmanica K. & S.S. Larsen
ปอเกี๋ยน Po kian (Northern).
B. penicilliloba Pierre ex Gagnep.
เสี้ยวแดง Siao dang (Loei).
B. pottisii G. Don var. pottsii
ชิงโค Ching kho (Ranong, Surat Thani); ชงโคดํา
Chong kho dam (Trang).
var. decipiens (Craib)
ชงโค Chong kho (Trat).
K. & S.S. Larsen
var. mollissima
ชงโคไฟ Chong kho fai (Penninsular).
(Wall. Ex Prain) K. & S.S. Larsen
var. subsessilis (Craib)
ชงโคขาว Chong kho khao (Central); ชงโคปา Chong
kho pa (Chanthaburi); ชัง่ โค Chang kho (Trat); ชิง
โค Ching kho, สมเสี้ยว Som siao (Surat Thani); ชุม
โค Chum kho (Chumphon).
var. velutina (Wall. Ex Benth.)
ชงโค Chong kho (Ranong).
K. & S.S. Larsen
B. pulla Craib.
กาหลง Kalong (Nakhon Sawan); แสลงพัน Saleang
phan (Nakhon Ratchasima); แสลงพันเถา Salaeng
phan thao (Nakhon Sawan).
B. purpurea L.
กะเฮอ Ka-hoe, สะเปซี
Sa-pe-si (Karen-Mae-
Hong-Son); ชงโค Chong kho (Central); เสี้ยวดอก
แดง Siao dok daeng (Northern); เสี้ยวหวาน Siao
wan (Mae Hong Son); Orchid tree, Purple
bauhinia.
B. racemosa Lam.
ชงโคขี้ไก Chong kho khi kai (Kanchanaburi); ชงโค
นา Chong kho na, ชงโคใบเล็ก Chong kho bai lek
(Ratchaburi); ชงโคเล็ก Chong kho lek (Saraburi);
5
สมเสี้ยว Som siao (Lampang); เสี้ยว Siao
(Northern); เสี้ยวใหญ Siao yai (Prachin Buri).
B. saccocalyx Pierre
คิงโค Khing kho (Nakhon Ratchasima); ชงโค
Chong kho (Chanthaburi, Nakhon Ratchasima,
Suphan Buri, Uthai Thani); สมเสี้ยว Som siao
(Nakhon Sawan, Udon Thani); สมเสีย้ วโพะ Som
siao po, เสี้ยวดอกขาว Siao dok khao; เสี้ยวปา Siao
pa (Nan).
santiwongsei Craib
= B. bassacensis Pierre ex Gagnep.
B. scandens L.
กระไดลิง Kradai ling (Ratchaburi); กระไดวอก
var horsfieldii (Miq.)
Kradai wok (Northern); โชกนุย Chok-nui
K. & S.S. Larsen
(Chaobon-Chaiyaphum); มะลืมดํา Ma luem dam
(Chaing Mai).
B. sirindhorniae
สามสิบสองประดง Sam sip song pra dong (Nong
K. & S.S. Larsen
Khai); สิรน
ิ ธรวัลลี Sirinthon wanly (Bangkok).
B. similes Craib.
แสลงพันกระดูก
Salaeng
phan
kraduk
(Kanchanaburi).
B. strychnifolia Craib
ขยัน Khayan, เครือขยัน Khruea khayan (Northern);
สยาน Sayan (Tak, Lampang); หญานางแดง Ya nang
daeng (Northeastern).
B. strychnoidea Prain
โชคนุย Chok Nui (Narathiwat).
subsessilis Craib
= B. pottsii G. Don var. subsessilis (Craib) de Wit
sulphurea Craib
= B. bassacensin Pierre ex Gagnep.
tenuiflora Watt ex C.B. Clarke
= B. glauca (Wall. Ex Benth.) Benth. Subsp.
tenuiflora (Watt ex C.B. Clarke) K. & S.S.
Larsen
B. tomentosa L.
ชงโคดอกเหลือง Chong kho dok lueang (Bangkok).
6
B. variegata L.
เปยงพะโก Piang phako (Sukhothai); โพะเพ Phophe (Karen-Kanchanaburi); เสี้ยวดอกขาว Siao dok
khao (Northern) นางอั้ว Nang ua (Chaing Mai);
Mountain ebony tree, St. Thomas tree.
velutina Wall. Ex Benth.
= B. pottsii G.Don var. velutina (Wall. Ex Benth.)
K. & S.S. Larsen
B. viridescens Desv.
var. viridescens.
บะหมะคอมี Ba-ma-kho-mi (Karen-Kanchnaburi);
สมเสี้ยวนอย Som siao noi (Prachin Buri); สมเสี้ยว
บาง Som siao bai bang (Prachuap Khiri Khan);
เสี้ยวเคี้ยว Siao khiao (Loei); เสี้ยวนอย Siao noi, เสี้ยว
ปอก Siao pok (Phrae); เสี้ยวฟอม Siao fom
(Northern).
var. hirsuta K. & S.S. Larsen
กาหลงเขา Kalong khao (Kanchanaburi).
B. wallichii J.F. Macbr.
ชงโคภูคา Chong kho phuka (Nan)
B. wintii Craib
คิว้ นาง Khio nang, อรพิม Ora phim (Central).
B. yunnanensis Franch.
เสี้ยวแพะ Siao phae (Lamphun); หญาเกล็ดปลามง Yaklet-pla-mong (Shan-Northern).
Bauhinia sirindhorniae K. & S.S. Larsen is an indigenous plant known in Thai
as Sirinthon Wanli or Sam Sip Song Pra Dong and is a trendrilled liana (Figure 1).
Young branches are hairy reddish brown and grabrous. Stipules are oblong-elliptic
and early caduceus. Leaves are coriaceous and ovate. The apex is slighty bifid to
deeply bifid almost to the base. Inflorescences are densely ferrugineous pubescent in
which bracts are hairy outside and glabrous inside.
Hypanthium is tubular to
narrowly funnel-shaped, striate and hairy. Calyx is splited on one side to the base and
on the opposite side at the tip only. Petals are densely hairy reddish brown. Stamens
are three fertile. The filaments and anthers are glabrous. There are two staminodes
with triangular and minute. Ovary is hairy reddish brown. Pods are ferrugineous
pubescent. Seeds are 5-7, dark brown, flat and orbicular (Larsen and Larsen, 1997).
7
The genus Croton belongs to the family Euphorbiaceae. They comprise of
approximately 700 species which distributed over all warm countries. Croton species
are reported to possess important medicinal uses and well known as toxic plants.
Most members are trees or shrubs and a few are herbs. Leaves are usually
alternate with biglandular at the base. The flowers are small bracts. Male flowers
contain five calyx, five petals and a disk of 4-6 glands opposite the sepals. There are
many stamens inserted on a hairy receptacle and the anthers are adnate with parallel
cells. In female flowers, sepals are usually more ovate than the male and the petals
are smaller than the sepals or missing. The disk is annular and consists of 4-6 glands
are opposite the sepals. There are three ovaries with solitary ovule in each cells,
styles are usually long and slender. Seeds are smooth, albumen copious and broad
cotyledon (Shaw 1972).
The species of genus Croton which have been recorded in Thailand
(Smitinand, 2001), are as follows:
จิมิจิยา Chi-mi-chi-ya, เปลา Plao, เปลาแพะ Plao
C. acutifolius Esser
phae, มะดอไก Mado kai (Northern).
เปลา Plao (Prachuap Khiri Khan); เปลาเงิน Plao
C. argyratus Blume
ngoen (Nong Khai).
birmanicus Müll.Arg.
=
C. tiglium L.
C. bonplandianus Daillon.
เปลาทุง Plao thung (General).
C. cascarilloides Raeusch.
เปลาเงิน Plao ngoen (Songkhla); เปลาน้ําเงิน Plao
nam ngoen (Prachuap Khiri Khan).
C. caudatus Geiseler
กระดอหดใบขน Krado hot bai khon (Chanthaburi);
โคคลาน Kho khlan (Nakhon Ratchasima); ปริก
Prik (Trang); โคคลาน ใบขน Kho khlan bai khon
(General);
กูเราะปริยะ
Ku-ro-pri-ya
(Malay-
Narathiwat).
C. columnaris Airy Shaw
เปลาคํา Plao Kham (Sukhothai).
C. crassifolius Geiseler
ปงคี Pang khi, พังคี Phang khi (Chiang Mai).
8
cumingii Müll. Arg.
=
C. cascarilloides Raeusch.
เปลา Plao, เปลานอย Plao noi, นมน้ําเขียว Nom nam
C. delpyi Gagnep.
khiao (Southeastern).
C. griffithii Hook. f.
จิก Chik, เปลา Plao (Peninsular).
C. hirtus L. Her.
เปลาลมลุก Plao lom luk (Peninsular).
C. hutchinsonianus Hosseus.
เปลา Plao, เปลาแพะ Plao phae, เปลาเลือด Plao lueat,
แมลาเลือด Mae la lueat, เหมือดฮอน Mueat hon
(Northern).
C. kerrii Airy Shaw
เปลา Plao (General).
C. kongensis Gagnep.
เปลาเงิน
Plao
(Northeastern);
ngoen,
เปลานอย
Plao
noi
เปลาน้ําเงิน Plao nam ngoen
(Eastern); เสปอตุ Se-po-tu (Karen–Chieng Mai).
ทรายขาว Sai Khao (Northern); พริกนา Prik na
C. krabas Gagnep.
(Central); ฝายน้ํา Fai nam (Eastern).
C. lachnocarpus Benth.
ขี้อน Khi on (Southwestern).
C. longissimus Airy Shaw
เปลานอย Plao noi (Lampang).
C. mekongensis Gagnep.
เปลาน้ําเงิน
Plao nam ngoen, พริกนา Prik na
(Northern).
oblongifolius Roxb.
=
C. cascarilloides Raeusch.
เปลา Plao, เปลาใหญ Plao yai (Southeastern); เปลา
C. poilanei Gagnep.
หลวง Plao luang, เปลาเลือด Plao lueat (Northern).
pierrei Gagnep.
=
เปลาเลือด Plao lueat (Lampang).
C. robustus Kurz
rottleri Geiseler
C. cascarilloides Raeusch.
=
Chrozophora rottleri (Geiseler) A. Juss ex
Spreng.
C. roxburghii N.P. Balakr.
ควะวู Khwa-wu (Karen–Kanchanaburi); เซงเคคัง
Seng–khe-khang, สะกาวะ Sa-ka-wa, สากูวะ Sa-ku-
9
wa (Karen-Mae Hong Son); เปาะ Po (Kamphaeng
Phet); เปลาหลวง Plao luang (Nortern); เปลาใหญ
Plao yai (Central); หาเยิ่ง Ha-yoeng (Shan-Mae
Hong Son).
C. santisukii Airy Shaw
เปลาสันติสุข Plao santisuk (Southwestern).
C. sepalinus Airy Shaw
เปลาเงิน Plao ngoen (Peninsular).
siamensis Craib
=
C. robustus Kurz
เปลานอย Plao noi (Prachin Buri, Prachuap Khiri
C. stellatopilosus Ohba
Khan); เปลาทาโพ Plao tha po (Southeastern).
C. thorelii Gagnep.
เปลาตะวัน Plao tawan (Southeastern).
C. tiglium L.
บะกั้ง Ba kang (Phrae) ; มะขาง Ma khang, มะคัง Ma
khang, มะตอด Matot, หมากทาง Mak thang, หัสคืน,
Has sa khuen (Northern); ลูกผลาญศัตรู Luk phlan
sattru, สลอด Salot, สลอดตน Salot ton, หมากหลอด
Mak lot (Central); หมากยอดง Mak-yong (ShanMae Hong Son); Croton oil plant.
tomentosus Müll.Arg.
C. trachycaulis Airy Shaw
=
C. crassifolius Geiseler
กวะวะ Kwa-wa, กวาโอะวะ Kwa-o-wa (KarenKanchanaburi); ขี้อน Khi on (Prachuap Khiri
Khan).
C. wallichii Müll.Arg.
เปลา Plao, เปลานา Plao na (General).
Croton hutchinsonianus Hosseus. has a local name as Plao phae (Figure 2). It
is a shrub or small tree reaching 4-5 m, locates commonly in dry mixed deciduous
forest or open scrub and grows on lateritic or sandstone soil. Bark is corky and
deeply cracked with a deep red sap. It is a coarse plant, with large coriaceous leaves
densely minutely steallate-pubescent. The inflorescence is densely whitish steallatetomentose (Shaw 1972).
10
During our preliminary evaluation for biological activities, the extract of
Bauhinia sirindhorniae showed significant scavenging activities towards 1,1diphenyl-2-picrylhydrazyl (DPPH) radical activity whereas Croton hutchinsonianus
showed significant cytotoxicity activity.
As for Bauhinia sirindhorniae, no
phytochemical work has been reported. Therefore, the following objectives are put
forwards:
1. To isolate and purify compounds from the stems and the roots of Bauhinia
sirindhorniae, and from the branches and the leaves of Croton
hutchinsonianus.
2. To determine the chemical structure of each isolated compound.
3. To evaluate the biological activities of each isolated compound.
11
Figure 1 Bauhinia sirindhorniae K. & S.S. Larsen.
12
Figure 2 Croton hutchinsonianus Hosseus.
CHAPTER II
HISTORICAL
1. Chemical Constituents of Bauhinia spp.
A number of compounds has been isolated from the genus Bauhinia. They are
classified as flavonoids, triterpenoids, steroids, cyanoglucosides, alkaloids, stibenes,
lignans, phenylpropanoids and miscellaneous substances (Table 1-3).
Table 1 Distribution of flavonoids in Bauhinia spp.
Plant and Chemical compound
Plant part
Reference
Bauhinia candidans
Kaempferol-3-O-β-rutinoside [1]
Leaf
Iribarren and Pomilio, 1983
Leaf
Iribarren and Pomilio, 1983
Root
Chen et al., 1984
OH
HO
O
O-Glucose-Rhamnose
OH
O
Kaempferol-3-O-β-rutinoside-7-Oα-rhamnopyranoside [2]
OH
Rhamnose-O
O
O-Glucose-Rhamnose
OH
O
B. championii
5,6,7,5′-Tetramethoxy-3′,4′methylene dioxyflavone [3]
O
O
MeO
O
OMe
MeO
OMe
O
14
Table 1 (continued)
Plant and Chemical compound
5,6,7,3′,4′,5′-Hexamethoxyflavone [4]
Plant part
Reference
Root
Chen et al., 1984
Root
Chen et al., 1984
Root
Chen et al., 1984
Root
Chen et al., 1984
Root
Chen et al., 1984
OMe
OMe
MeO
O
OMe
MeO
OMe
O
5,7,5′-Trimethoxy-3′,4′-methylene
dioxyflavone [5]
O
O
MeO
O
OMe
OMe
O
5,6,7,3′,4′-Pentamethoxyflavone [6]
OMe
OMe
MeO
O
MeO
OMe
O
5,7,3′,4′,5′-Pentamethoxyflavone [7]
OMe
OMe
MeO
O
OMe
OMe O
5,7,3′,4′-Tetramethoxyflavone [8]
OMe
OMe
MeO
O
OMe
O
15
Table 1 (continued)
Plant and Chemical compound
Plant part
Reference
B. guianensis
4′-Hydroxy-7-methoxyflavan [9]
Stem bark
Viana et al., 1999
OMe
O
HO
B. manca
Stem
Apigenin [10]
Achenbach, Stocker and
Constenla, 1988
OH
O
OH
OH
O
Chrysoeriol [11]
Stem
Achenbach et al., 1988
Stem
Achenbach et al., 1988
Stem
Achenbach et al., 1988
OMe
OH
HO
O
OH
O
Luteolin-5,3′-dimethyl ether [12]
OMe
HO
O
OH
OMe
O
Kaempferol [13]
OH
HO
O
OH
OH
O
16
Table 1 (continued)
Plant and Chemical compound
Isoliquiritigenin [14]
Plant part
Reference
Stem
Achenbach et al., 1988
Stem
Achenbach et al., 1988
Stem
Achenbach et al., 1988
Stem
Achenbach et al., 1988
Stem
Achenbach et al., 1988
Stem
Achenbach et al., 1988
OH
HO
OH
O
(2S)-Liquiritigenin [15]
OH
HO
O
O
(2S)-Eriodictyol [16]
OH
OH
HO
O
OH
O
(2S)-Naringenin [17]
H
OH
HO
O
OH
O
Isoliquiritigenin-2′-methyl ether [18]
OH
HO
OMe
O
Isoliquiritigenin-4-methyl ether [19]
OMe
HO
OH
O
17
Table 1 (continued)
Plant and Chemical compound
Echinatin [20]
Plant part
Reference
Stem
Achenbach et al., 1988
Stem
Achenbach et al., 1988
Stem
Achenbach et al., 1988
Stem
Achenbach et al., 1988
Stem
Achenbach et al., 1988
Stem
Achenbach et al., 1988
Stem
Achenbach et al., 1988
OH
HO
OMe
O
(2S)-Liquiritigenin-7-methyl ether [21]
OH
MeO
O
O
(2S)-Liquiritigenin-4′-methyl ether [22]
OMe
HO
O
O
(2S)-7,4′-Dihydroxyflavan [23]
OH
HO
O
(2S)-4′-Hydroxy-7-methoxyflavan [24]
OH
MeO
O
(2S)-7,3′-Dimethoxy-4′-hydroxy
flavan [25]
OH
MeO
O
OMe
(2S)-3′,4′-Dihydroxy-7-methoxy
flavan [26]
OH
MeO
O
OH
18
Table 1 (continued)
Plant and Chemical compound
(2S)-7,4′-Dihydroxy-3′-methoxy
Plant part
Reference
Stem
Achenbach et al., 1988
Stem
Achenbach et al., 1988
Stem
Achenbach et al., 1988
Stem
Yadava and Tripathi, 2000
Wood
Laux Stefani and Gottlieb,
flavan [27]
OH
HO
O
OMe
Obtustyrene [28]
HO
OMe
2,4′-Dihydroxy-4-methoxy
dihydrochalcone [29]
OH
MeO
OH
O
B. purpurea
5,6-Dihydroxy-7-methoxyflavone-6O-β-D-xylopyranoside [30]
MeO
O
Xylos-O
OH
O
Bausplendin [31]
1985
O
O
MeO
O
O
O
O
Chrysin [32]
Bark
O
OH
OH
O
Kuo, Chu and Chang, 1998
19
Table 1 (continued)
Plant and Chemical compound
6,8-Dimethylchrysin [33]
Plant part
Reference
Bark
Kuo et al., 1998
Stem
Gupta, Vidyapati and
O
OH
OH
O
B. variegata
Naringenin-5,7-dimethylether-4′-
Chauhan, 1980
rhamnoglucoside [34]
O-Glucose-Rhamnose
O
MeO
OMe
O
20
Table 2 Distribution of steroids in Bauhinia spp.
Plant and Chemical compound
Plant part
Reference
B. candidans
Campesterol [35]
Leaf
Iribarren and Pomilio, 1983
Leaf
Iribarren and Pomilio, 1983
Leaf
Iribarren and Pomilio, 1983
Leaf
Iribarren and Pomilio, 1983
Leaf
Iribarren and Pomilio, 1983
HO
Cholesterol [36]
HO
Daucosterol [37]
Glucose-O
β-Sitosterol [38]
HO
Stigmasta-3,5-dien-7-one [39]
O
21
Table 2 (continued)
Plant and Chemical compound
B. candidans
Plant part
Reference
Aerial part
Iribarren and Pomilio, 1983
Stem bark
Viana et al., 1999
Sitosterol-3-O-α-D-riburono
furanoside [40]
Ribose-O
B. guianensis
Stigmasta-5,22-dien-3-O-β-Dglucopyranoside [41]
Glucose-O
B. manca
Stigmasta-4-en-3,6-dione [42]
Stem
Achenbach et al., 1988
Stem
Achenbach et al., 1988
O
O
Stigmasta-4-en-3-one [43]
O
22
Table 2 (continued)
Plant and Chemical compound
Plant part
Reference
B. purpurea
Stigmasta-5-en-7-one-3-O-β-D-
Bark
Kuo et al., 1998
Bark
Kuo et al., 1998
glucopyranoside [44]
Glucose-O
O
6′-(Stigmasta-5-en-7-one-3-O-β-Dglucopyranosidyl) hexadecanoate [45]
Hexadecanoate-Glucose-O
O
B. uruguayensis
Stigmasta-1,3,5-triene [46]
Aerial part
Iribarren and Pomilio, 1989
Stigmasta-3,5-diene [47]
Aerial part
Iribarren and Pomilio, 1989
23
Table 2 (continued)
Plant and Chemical compound
Stigmasterol [48]
HO
Plant part
Reference
Aerial part Iribarren and Pomilio, 1989
Aerial part Iribarren and Pomilio, 1989
Stigmasta-4,6-dien-3-one [49]
O
Sitosterol-3-O-β-D-xylopyranoside [50]
Aerial part Iribarren and Pomilio, 1989
Xylose-O
Sitosterol-3-O-α-D-xylurono
Aerial part Iribarren and Pomilio, 1989
furanoside [51]
Xylose-O
Sitosterol-3-O-β-D-glucopyranoside [52]
Glucose-O
Aerial part Iribarren and Pomilio, 1989
24
Table 3 Distribution of miscellaneous compounds in Bauhinia spp.
Plant and Chemical compound
Category
Plant part
Cyanoglucoside
Root
Reference
B. championii
Bauhinin [53]
NC
Glucose
Chen, Chen and
Hsu, 1985
O
HO
OMe
B. fassoglensis
Lithospermoside [54]
Cyanoglucoside
Root
NC
Glucose
Fort, Jolad and
Nelson, 2001
O
HO
OH
B. guianensis
Lapachol [55]
Quinoid
Stem bark
Stilbene
Root
Viana et al., 1999
O
OH
O
B. malabarica
Preracemosol A [56]
Kittakoop et al.,
2000
OMe
HO
HO
OH
25
Table 3 (continued)
Plant and Chemical compound
Preracemosol B [57]
Category
Plant part
Reference
Stilbene
Root
Kittakoop et al., 2000
Lignan
Stem
Achenbach et al., 1988
Lignan
Stem
Achenbach et al., 1988
Benzenoid
Stem
Achenbach et al., 1988
Phenyl
Stem
Achenbach et al., 1988
O
HO
HO
OH
B. manca
(7S,8R,8′R)-5-5′-Dimethoxy
lariciresinol [58]
OMe
HO
O
MeO
H
H
OMe
OH
OH
OMe
Syringaresinol [59]
OMe
HO
O
MeO
H
H
OMe
O
OH
OMe
Gallic acid [60]
COOH
HO
HO
OH
Cinnamic acid [61]
O
OH
propanoid
26
Table 3 (continued)
Plant and Chemical compound
Cinnamoyl-β-D-glucoside [62]
O
Category
Plant part
Phenyl
Stem
propanoid
Reference
Achenbach et al.,
1988
O-Glucose
ω-Hydroxypropioguaiacone [63]
CH2CH2OH
C
Phenyl
Stem
propanoid
Achenbach et al.,
1988
O
OMe
OH
B. racemosa
Racemosol [64]
Stilbene
Heartwood
Anjaneyulu, Reddy
and Reddy, 1986
O
OH
HO
OH
De-O-methylracemosol [65]
Stilbene
Root
Prabhakar et al.,
1994
O
OMe
HO
OH
Stilbene
Pacharin [66]
Heartwood
HO
Anjaneyulu et al.,
1984
O
OMe
HO
Resveratrol [67]
Stilbene
OH
HO
OH
Heartwood
Anjaneyulu et al.,
1984
27
Table 3 (continued)
Plant and Chemical compound
Category
Plant part
Reference
Stilbene
Root bark
Millard et al., 1991
Stilbene
Root bark
Millard et al., 1991
Stilbene
Root bark
Millard et al., 1991
Cyclohexenone
Leaf
B. rufescens
5,6-Dihydro-11-methoxy-2,2,12trimethyl-2H-naphthol[1,2-f][1]
benzopyran-8,9-diol [68]
MeO
O
HO
HO
11-Methoxy-2,2,12-trimethyl2H-naphthol[1,2f][1]benzopyran-8,9-diol [69]
MeO
O
HO
HO
1,7,8,12b-Tetrahydro-2,2,4trimethyl-2H-benzo[6,7]
cyclohepta[1,2,3-de][1]
benzopyran-5,10,11-triol [70]
O
OH
HO
OH
B. tarapotensis
2,4-Dihydroxy-2-(2-hydroxy
ethyl) cyclohexe-5-en-1-one [71]
OH
HOH2CH2C
OH
O
Braca et al., 2001
28
Table 3 (continued)
Plant and Chemical compound
Indole-3-carboxylic acid [72]
Category
Plant part
Reference
Alkaloid
Leaf
Braca et al., 2001
Lignan
Leaf
Braca et al., 2001
Lignan
Leaf
Braca et al., 2001
Phenyl
Leaf
Braca et al., 2001
Leaf
Braca et al., 2001
COOH
N
H
(-)-Isolariciresinol-3-α-O-β-Dglucopyranoside [73]
CH 2 OH
MeO
HO
CH 2 -O-Glucose
OMe
OH
(+)-1-Hydroxypinoresinol-1-O-β-Dglucopyranoside [74]
OMe
OH
O
O-Glucose
H
O
HO
OMe
Isoacteoside [75]
Propanoid
HO
COOCH2
HO
Glucose
OH
OCH2CH2
OH
Glucose
Caffeoyl ester of apionic acid [76]
HO
HO
COO CH2
CH
CH
COHCH2OH
CHOH
COOH
Phenyl
propanoid
29
Table 3 (continued)
Plant and Chemical compound
B. variegata
Lupeol [77]
HO
Category
Plant part
Triterpene
Stem
Reference
Gupta et al., 1980
30
2. Chemical Constituents of Croton spp.
Chemical investigations of a number of Croton species have been shown to be
a good source of diterpenes. In addition, other classes of natural compounds such as
flavonoids, alkaloids, monoterpenes, triterpenes and miscellaneous substances have
been found (Tables 4-7).
Table 4 Distribution of diterpenes in Croton spp.
Plant and Chemical compound
Plant part
Reference
Croton argyrophylloides
3,12-Dioxo-15,16-epoxy-4-hydroxy
Trunk wood
cleroda-13(16),14-diene [78]
Monte, Dantas and Braz,
1988
O
O
O
HO
ent-Kaur-16-en-15-oxo-18-oic acid
Trunk wood
Monte et al., 1988
Trunk wood
Monte et al., 1984
Trunk wood
Monte et al., 1984
[79]
O
HOOC
Tetracyclic diterpenic acid [80]
HOOC
O
O
Tetracyclic diterpene ester [81]
MeOOC
O
O
31
Table 4 (continued)
Plant and Chemical compound
Plant part
Reference
C. aromaticus
(-)-Hardwickiic acid [82]
Root
Bandara, Wimalasiri and
Bandara, 1987
O
COOH
C. cajucara
Cajucarins A [83]
Bark
Itokawa et al., 1990
Bark
Itokawa et al., 1990
Bark
Ichihara et al., 1991
Bark
Ichihara et al., 1991
O
H
COOMe
O
CHO
Cajucarins [84]
O
COOMe
O
Cajucarinolide [85]
HO
O
O
O
O
H
O
H
Isocajucarinolide [86]
O
O
OH
O
H
O
O
H
32
Table 4 (continued)
Plant and Chemical compound
trans-Crotonin [87]
Plant part
Reference
Bark
Itokawa et al., 1989
Bark
Itokawa et al., 1989
Bark
Kubo, Asaka and Shibata,
O
O
O
H
O
H
Dehydrocrotonin (transdehydrocrotonin) [88]
O
O
O
H
O
H
cis-Dehydrocrotonin [89]
O
O
1991
O
H
O
H
trans-Cajucarin [90]
O
O
H
Bark
Maciel et al., 1998
Bark
Maciel et al., 1998
COOMe
H
Sacacarin [91]
O
O
H
O
O
33
Table 4 (continued)
Plant and Chemical compound
Plant part
Reference
C. californicus
(-)-Methyl barbascoate [92]
O
Leaf and terminal
branch
Wilson, Neubert and
Huffman, 1976
O
COOMe
C. campestris
Velamone [93]
Root
Babili et al., 1997
Root
Babili et al., 1997
Root
Babili et al., 1997
O
O
H
Velamolone [94]
O
O
H
CH2OH
Velamone acetate [95]
O
O
H
CH2OAc
34
Table 4 (continued)
Plant and Chemical compound
Plant part
Reference
C. caudatus
Crotocaudin [96]
Stem bark
1977
O
O
H
O
Chatterjee and Banerjee,
O
H
H
O
O
Stem bark
Isocrotocaudin [97]
1977
O
O
H
O
Chatterjee and Banerjee,
O
H
H
O
O
Stem bark
Teucvidin [98]
Chatterjee and Banerjee,
1977
O
H
O
H
O
O
H
O
O
Stem bark
Teucin [99]
O
H
O
H
O
O
H
O
O
Chatterjee and Banerjee,
1977
35
Table 4 (continued)
Plant and Chemical compound
Plant part
Reference
C. cortesianus
Hoffmanniaaldehyde [100]
Aerial part
Seims et al., 1992
Aerial part
Seims, Dominguez and
O
H
CHO
5,10-Dihydro-5α-hydroxy-10βprintziane [101]
Jakupovic, 1992
O
H
Stigillanoic acid B [102]
Aerial part
Seims et al., 1992
O
H
COOH
C. corylifolius
Leaf and twig
Corylifuran [103]
O
H
H
O
O
COOMe
COOMe
Burke, Chan and Pascoe,
1979
36
Table 4 (continued)
Plant and Chemical compound
Crotofolin A [104]
Plant part
Reference
Leaf and twig
Burke et al., 1979
Leaf and twig
Burke et al., 1979
Leaf and twig
Burke et al., 1979
Leaf and twig
Burke et al., 1979
H
O
O
H
HO
H
OH
Crotofolin B [105]
H
O
O
H
H
HO
OH
Crotofolin C [106]
O
O
H
H
Crotofolin E [107]
OH
O
O
H
O
H
O
C. crassifolius
Chettaphanin-I [108]
Root
O
Boonyaratanakornkit et al.,
1988
O
O
OH
COOMe
Cyperenoic acid [109]
Root
Boonyaratanakornkit et al.,
1988
HOOC
37
Table 4 (continued)
Plant and Chemical compound
Plant part
Reference
C. diasii
Diasin [110]
Trunk wood
Alvarenga et al., 1978
Trunk wood
Alvarenga et al., 1978
O
H
O
H
H
H
O
H
O
O
OMe
O
Isodiasin [111]
O
H
O
H
H
H
O
H
O
O
OMe
O
C. dichigamus
Crotoxide A [112]
Leaf
Jogia and Anderson, 1989
Leaf
Jogia and Anderson, 1989
HO
O
O
O
Crotoxide B [113]
O
O
H
H
H
O
O
H
O
C. eluteria
Stem bark
Cascarillin B [114]
O
H
O
O
O
O
O
Vigor et al., 2001
38
Table 4 (continued)
Plant and Chemical compound
Cascarillin C [115]
Plant part
Reference
Stem bark
Vigor et al., 2001
Stem bark
Vigor et al., 2001
Stem bark
Vigor et al., 2001
Stem bark
Vigor et al., 2001
Stem bark
Vigor et al., 2001
O
O
OH
H
O
O
Cascarillin D [116]
O
H
O
O
O
O
Cascarillin E [117]
O
O
OMe
H
H
HO
O
HO
O
Cascarillin F [118]
O
O
OH
H
H
HO
O
HO
O
Cascarillin G [119]
O
H
HO
O
OH
H
O
HO
OCOMe
O
39
Table 4 (continued)
Plant and Chemical compound
Plant part
Reference
Trunk bark
Tchissambou et al., 1990
Trunk bark
Tchissambou et al., 1990
Stem bark
Krebs and Ramiarantosa,
C. haumanianus
Crotocorylifuran [120]
O
H
O
O
H
COOMe
COOMe
Crotohaumanoxide [121]
O
O
H
H
H
O
O
H
O
C. hovarum
3α,4β-Dihydroxy-15,16-epoxy-12-oxo-
1996
cleroda-13(16),14-diene [122]
O
H
O
HO
HO
3α,4β-Dihydroxy-15,16-epoxy-12-oxocleroda-13(16),14-diene-9-al [123]
O
H
HO
HO
O
CHO
Stem bark
Krebs and Ramiarantosa,
1996
40
Table 4 (continued)
Plant and Chemical compound
Plant part
Reference
C. joufra
Leaf
2α,3α-Dihydroxy-labda-
Sutthivaiyakit et al.,
2001
8(17),12(13),14(15)-triene [124]
HO
HO
3β-Hydroxy-19-O-acetyl-pimara-8(9),15Leaf
diene-7-one [125]
Sutthivaiyakit et al.,
2001
O
HO
OCOMe
C. kerrii
(E,E,Z)-11-Hydroxymethyl-3,7,15-trimethyl
Leaf
-2,6,10,14-hexadecatetraen-1-ol [126]
Sato, Ogiso and
Kuwano, 1980
CH2OH
OH
(E,E,E)-11-Formyl-3,7,15-trimethyl2,6,10,14-hexadecatetraen-1-ol [127]
Leaf
Sato et al., 1980
Leaf
Thongtan et al., 2003
CHO
OH
C. kongensis
ent-8,9-seco-7α,11β-Diacetoxykaura8(14),16-dien-9,15-dione [128]
O
O
H
O
O
OAc
41
Table 4 (continued)
Plant and Chemical compound
Plant part
Reference
ent-8,9-seco-8,14-Epoxy-7α-hydroxy-11β-
Leaf
Thongtan et al., 2003
Leaf
Thongtan et al., 2003
Root
Bandara, Wimalasiri
acetoxykaura-16-kauren-9,15-dione [129]
O
O
H
O
O
O
OH
ent-8,9-seco-7α,11β-Diacetoxykaura8(14),16-dien-9,15-dione [130]
O
O
H
O
O
OH
C. lacciferus
16α-H-ent-Kauran-17-oic acid [131]
and Macleod, 1988
H
COOH
ent-Kaur-15-en-3β,17-diol [132]
Root
Bandara et al., 1988
Root
Bandara et al., 1988
CH2OH
HO
ent-Kaur-15β,16-epoxykauran-17-ol [133]
CH2OH
O
HO
42
Table 4 (continued)
Plant and Chemical compound
Plant part
Reference
C. lechleri
Bincatriol [134]
Bark
Chen, Cai and
OH
OH
Phillipson, 1994
HO
Crolechinic acid [135]
OH
Bark
Chen et al., 1994
Bark
Chen et al., 1994
Bark
Chen et al., 1994
Leaf and twig
Chan, Taylor and
OH
H
COOH
Korberin A [136]
O
H
H
O
MeOOC
O
O
COOMe
Korberin B [137]
O
H
H
O
O
COOMe
COOMe
C. lucidus
Crotonin [138]
O
O
O
O
Willis, 1968
43
Table 4 (continued)
Plant and Chemical compound
Plant part
Reference
C. macrostachys
Seed
Crotomachlin [139]
Herlem, Huu and
Kende, 1993
OH
H
OH
H
OH
Neoclerodan-5,10-en-19,6β;20,12diolide [140]
Root
Kapingu et al., 2000
Root
Kapingu et al., 2000
Root
Kapingu et al., 2000
Root
Kapingu et al., 2000
Root
Kapingu et al., 2000
O
O
O
O
O
O
3α,19-Dihydroxytrachylobane [141]
HO
CH2OH
3α,18,19-Trihydroxytrachylobane [142]
HO
HOH2C
CH2OH
Trachyloban-19-oic acid [143]
H
COOH
Trachyloban-18-oic acid [144]
H
HOOC
44
Table 4 (continued)
Plant and Chemical compound
Plant part
Reference
C. megalocarpus
Criromodine [145]
Bark
Mensah et al., 1989
Bark
Mensah et al., 1989
Leaf
Jas and Hahn, 1978
O
O
O
H
OMe
OH
HO
Epoxychiromodine [146]
O
O
O
H
OMe
O
C. niveus
Nivenolide [147]
O
O
H
HOOC
C. nitens
Leaf and twig
Crotonitenone [148]
Burke, Chan and
Pascoe, 1981
O
H
HO
H
O
C. oblongifolius
Crotocembraneic acid [149]
COOH
Stem bark
Vilaivan et al., 1997
45
Table 4 (continued)
Plant and Chemical compound
Neocrotocembraneic acid [150]
Plant part
Reference
Stem bark
Vilaivan et al., 1997
Stem bark
Roengsumran et al., 1999b
Stem bark
Aiyar and Seshadri, 1972
Stem bark
Aiyar and Seshadri, 1972
Stem bark
Aiyar and Seshadri, 1972
Stem bark
Roengsumran et al., 1999a
COOH
Neocrotocembranal [151]
CHO
11-Dehydro-(-)-hardwickiic acid [152]
H
O
COOH
ent-Isopimara-7,15-diene [153]
H
H
ent-Isopimara-7,15-diene-19-aldehyde
[154]
H
OHC
H
Labda-7,12(E),14-triene [155]
46
Table 4 (continued)
Plant and Chemical compound
Labda-7,12(E),14-triene-17-al [156]
Plant part
Reference
Stem bark
Roengsumran et al., 1999a
Stem bark
Roengsumran et al., 1999a
Stem bark
Roengsumran et al., 1999a
Stem bark
Aiyar and Seshadri, 1970
Stem bark
Aiyar and Seshadri, 1970
Stem bark
Aiyar and Seshadri, 1972
CHO
Labda-7,12(E),14-triene-17-ol [157]
CH2OH
Labda-7,12(E),14-triene-17-oic acid
[158]
COOH
Oblongifoliol [159]
H
HO
HOH2C
H
Oblongifolic acid [160]
H
HOOC
H
3-Deoxyoblongifoliol [161]
H
HOH2C
H
47
Table 4 (continued)
Plant and Chemical compound
Plant part
Stem bark
19-Deoxyoblongifoliol [162]
Reference
Aiyar and Seshadri, 1970
H
HO
H
C. poilanei
Poilaneic acid [163]
O
Leaf
Sato et al., 1981
Twig
Itokawa et al., 1991
Twig
Itokawa et al., 1991
Twig
Itokawa et al., 1991
OH
C. salutaris
(10E)-3,12-Dihydroxy-3,7,11,15tetramethyl-1,10,14-hexadecatrien-5,13dione [164]
O
O
OH
OH
(6E,10E)-3,12-Dihydroxy-3,7,11,15tetramethyl-1,6,10,14-hexadecatrien5,13-dione [165]
O
O
OH
OH
(6Z,10E)-3,12-Dihydroxy-3,7,11,15tetramethyl-1,6,10,14-hexadecatrien5,13-dione [166]
O
OH
O
OH
48
Table 4 (continued)
Plant and Chemical compound
12-Hydroxy-13-methylpodocarpa-9,
Plant part
Twig
Reference
Itokawa et al., 1991
11,13-trien-3-one [167]
OH
O
H
C. sonderianus
Sonderianol [168]
Heartwood
Craveiro and Silveira, 1982
Heartwood
Craveiro et al., 1981b
OH
O
H
Sonderianin [169]
O
O
O
COOMe
12-Hydroxyhardwickiic acid [170]
Root
McChesney, Clarke and
Silveira, 1991
O
OH
COOH
6α,7β-Dihydroxyannonene [171]
O
OH
OH
Root
Silveira and McChesney, 1994
49
Table 4 (continued)
Plant and Chemical compound
6α,7β-Diacetoxyannonene [172]
Plant part
Reference
Root
Silveira and McChesney, 1994
Stem
Ogiso et al., 1978
Stem
Kitazawa et al., 1979
Stem
Kitazawa et al., 1979
Stem
Kitazawa et al., 1980
O
OAc
OAc
C.sublyratus
Plaunotol [173]
CH2OH
CH2OH
Plaunol A [174]
O
H
O
H
H
O
H
OH
H
O
O
Plaunol B [175]
O
H
O
O
H
OH
H
O
O
Plaunol C [176]
O
H
O
HO
O
H
OH
O
O
H
50
Table 4 (continued)
Plant and Chemical compound
Plaunol D [177]
Plant part
Reference
Stem
Kitazawa et al., 1980
Stem
Kitazawa et al., 1980
Stem
Takahashi et al., 1983
Leaf
Block et al., 2004
Leaf
Block et al., 2004
O
OH
H
H OH
O
H
H
O
O
Plaunol E [178]
O
OAc
H
H OH
O
H
H
O
O
Plaunolide [179]
O
H
O
H
O
O
O
C. zambesicus
ent-18-Hydroxy-trachyloban-3-one
[180]
H
O
H
CH2OH
ent-Trachyloban-3-one [181]
H
O
H
51
Table 5 Distribution of triterpenes in Croton spp.
Plant and Chemical compound
Plant part
Reference
Croton cajucara
Acetyl aleuritolic acid [182]
Bark
Maciel et al., 1998
COOH
O
O
H
C. caudatus
Stem bark
Taraxerol [183]
Chatterjee and
Banerjee, 1977
H
H
HO
H
H
Taraxenone [184]
Stem bark
Chatterjee and
Banerjee, 1977
H
H
O
H
Taraxeryl acetate [185]
Stem bark
Chatterjee and
Banerjee, 1977
H
H
AcO
H
H
C. lacciferus
3β-Acetoxy-D-friedolean-14-en-28-oic
acid [186]
H
AcO
H
COOH
Root
Bandara et al., 1998
52
Table 6 Distribution of alkaloids in Croton spp.
Plant and Chemical compound
Plant part
Reference
Croton hemiargyreus
Hemiargyrine [187]
Leaf and stem
Amaral and Barnes, 1998
Leaf and stem
Amaral and Barnes, 1998
Leaf and stem
Amaral and Barnes, 1998
MeO
N
HO
H
OMe
OH
Glaucine [188]
OMe
MeO
MeO
MeO
N
H
Oxoglaucine [189]
OMe
MeO
MeO
MeO
N
O
C. lechleri
Taspine [190]
Leaf
Dennis et al., 2002
Leaf
Dennis et al., 2002
MeO
O N
O
O
O
OMe
Thaliporphine [191]
MeO
N
MeO
HO
OMe
53
Table 6 (continued)
Plant and Chemical compound
Plant part
Reference
C. membranaceus
Stem
Julocrotine [192]
Aboagye et al., 2000
CH2-CH2-Ph
N
O
O
O
Et
HC
C HN
C. salutaris
N-norsalutaridine [193]
Leaf and twig
HN
O
MeO
OH
Roderick and Orlando, 1981
54
Table 7 Distribution of miscellaneous compounds in Croton spp.
Plant and Chemical compound
Category
Plant part
Lignan
Stem
Reference
C. erythrochilus
4-O-Methyldihydrodehydro
Pieters , Dirk and
Arnold, 1990
diconiferyl alcohol [194]
OMe
OMe
O
OMe
HO(CH2)3
CH2OH
C. essequiboensis
Anethole [195]
Phenyl
OH
Leaf
Craveiro et al.,
1981a
propanoid
OH
Craveiro et al.,
1981a
propanoid
Phenyl
Estragole [196]
Leaf
CH2CH=CH2
β-Caryophyllene [197]
Sesquiterpene
Leaf
Craveiro et al.,
1981a
H
H
α-Copaene [198]
Sesquiterpene
Leaf
Craveiro et al.,
1981a
α-Cubenene [199]
Sesquiterpene
Leaf
Craveiro et al.,
1981a
H
55
Table 7 (continued)
Plant and Chemical compound
α-Humulene [200]
Category
Plant part
Sesquiterpene
Leaf
Reference
Craveiro et al.,
1981a
α-Pinene [201]
Monoterpene
Leaf
Craveiro et al.,
1981a
β-Pinene [202]
Monoterpene
Leaf
Craveiro et al.,
1981a
C. flavens
Phenanthrene
Crotoflavol [203]
Leaf
Wolfram and Franz,
2001
OH
MeO
OH
OMe
C. macrostachys
Crotepoxide [204]
Cyclohexane
Fruit
Kupchan,
O
O
diepoxide
CH2O-C-C6H5
Hemingway and
OAc
Smith, 1969
OAc
O
C. oblongifolius
Flavonoid
Isorhamnatin [205]
OMe
O
OH
OH
O
Subramanian,
Nagarajan and
OH
HO
Leaf
Sulochana, 1971
56
Table 7 (continued)
Plant and Chemical compound
Quercetin [206]
OH
O
OH
OH
O
Plant part
Flavonoid
Leaf
Reference
Subramanian et al.,
1971
OH
HO
Category
57
3. Literature reviews of Croton hutchinsonianus
In 1990, Chaoming et al. reported the presence of three benzenoid compounds,
(protocatechuic acid [207], methyl orsellinate [208], methyl 2,4-dihydroxy-3,6dimethylbenzoate [209]), two diterpenes (ent-kauran-16β,17diole [210], and entkauran-16β,17,19-triol [211]) , two steroids (β-sitosterol [38], β-sitosterol-Dglucoside
[37])
and
two
miscellaneous
compounds
(triacontanol
[212],
dotriacontanoic acid [213]) from the stem bark of C. hutchinsonianus (Chaoming et
al., 1990).
COOH
HO
HO
COOMe
OH
OH
[207]
COOMe
OH
OH
[208]
[209]
OH
OH
H
H
R
[210] R = Me
[211] R = CH2OH
HO
(CH2)29
[212]
Me
HOOC
(CH2)30
[213]
Me
58
4. Biosynthetic Relationship of Flavonoids in Bauhinia spp.
Flavonoids possess fifteen carbons atom in their basic skeletons, which are
derived from shikimate and acetate-malonate pathway. The typical flavonoids in
Bauhinia spp. are chalcone, flavone, flavanone, flavonol, dihydroflavonol and flavan.
The relationship of flavonoids is displayed in Scheme 1 (Markham, 1982).
Skimate pathway
OH
Acetate-malonate pathway
Cinnamyl alcohols
HOOC
Lignin
OH
OH
OH
O
HO
OH
HO
OH
O
O
(-)-Flavanone
Chalcone
HO
O
CH
OH
O
OH
Aurone
OH
OH
HO
OH
O
HO
OH
O
HO
OH
OH
OH
O
OH
Isoflavone
O
OH
(+)-Dihydroflavonol
O
OH
OH
(+)-Catechin
OH
+
O
HO
OH
(-)-Epicatechin
O
Flavonol
OH
OH
O
Flavone
OH
OH
O
OH
O
HO
OH
HO
O
HO
OH
HO
O
Dihydrochalcone
OH
OH
OH
Anthocyanidin
Scheme 1 Currently proposed interrelationships between flavonoid monomer.
59
5. Biosynthetic Relationship of Diterpenoids in Croton spp.
The diterpenes possess twenty carbon atoms in their molecules. They are
biogenetically derived from geranylgeranyl pyrophosphate (GGPP). The diterpene
skeleton is the fascinating variation encountered in their core structure, these
compounds could be classified into several types, such as mono-, bi-, tri-, tetra- and
pentacyclic diterpenes. The typical diterpenes in Croton spp. are casbane, cembrane,
clerodane, cleistanthane, kaurane, labdane, pimarane and halimane. The relationship
of diterpenes is displayed in Scheme 2. In addition, the biosynthetic is also proposed
(Devon and Scott, 1972).
OPP
Geranylgeranyl pyrophosphate
Cembrane
Rearrangement
Pimarane
Cleistanthane
Labdane
Stachane
Halimane
Clerodane
O
Kaurane
Seco-kaurane
Scheme 2 Biosynthetic relationship of diterpenes in Croton spp.
60
6. Traditional Uses and Biological Activities of Bauhinia spp.
Many plants of the genus Bauhinia have been used in traditional medicine in
several countries. The decoction of B. racemosa leaves has been used in the treatment
of headache and malaria, and its bark as an astringent for diarrhea and dysentery in
Indian medicine (Anjaneyulu et al., 1984). The one handful of grated stem bark of B.
guianensis is boiled in two liters of water until reduced to 1 liter, then drink half a cup
three times per day for stomachache and diarrhea (Mùnoz et al., 2000). In Nigeria,
the leaves of B. thonningii are used to treat diarrhea and fever (Kudi et al., 1999). B.
splendens is a native plant widely distributed in Brazil, being popularly known as
“cipo escada”, “cipo unha de boi”, “escada de jaboti” and “escada demacaco”. The
leaf and stem bark have been used as traditional remedies in folk medicine for the
management of several diseases, e.g. infections, inflammatory processes, diabetes and
infections of the urinary tracts (Filho et al., 1997). In Argentina and southern Brazil,
the infusion of B. candidans leaves is widely used because of theirs potential
hypoglycemic action (Irribarren and Pomilio, 1983).
A famous Thai traditional medicine from B. sirindhorniae is known as “Sam
Sip Song Pra Dong”. The infusion of its stem has been used as anti-inflammatory.
A number of biological investigations of Bauhinia species has been reported.
The 70% ethanol extract of B. guianensis was reported to possess antimalarial activity
(Mùnoz et al., 2000). The 80% ethanol extract of B. thonnigii showed inhibitory
effects against parvovirus (Kudi and Myint, 1999), gram-positive bacteria
Staphylococcus aureus, and gram-negative bacteria Escherichia coli (Kudi et al.,
1999). The 50% ethanol extract of B. splendens had a significant analgesic action
when assessed against several models of pain.
The mechanism underlying its
analgesic effect still remains unknown, but seems to be unrelated to interaction with
opioid systems (Filho et al., 1997).
The antimalarial activities of preracemosol A [56], preracemosol B [57],
racemosol [64] and demethylracemosol [65] from B. malabarica exhibited moderate
activities.
While only racemosol and demethylracemosol exhibited cytotoxicity
against KB and BC cell lines (Kittakoop et al., 2000).
As the root bark dichloromethane extract of B. rufescens showed antifungal
activity in a bioassay with the plant pathogenic fungus Cladosporium cucumerinum, a
phytochemical investigation was undertaken on material collected in Nigeria.
Activity
guided
fractionation
of
this
extract,
using
different
preparative
61
chromatographic methods, allowed the isolation of four antifungal tetracyclic
compounds: racemosol [64], 5,6-dihydro-11-methoxy-2,2,12-trimethyl-2H-naphthol
[1,2-f][1]benzopyran-8,9-diol [68], 11-methoxy-2,2,12-trimethyl-2H-naphthol[1,2f][1]benzopyran-8,9-diol
[69]
and
1,7,8,12b-tetrahydro-2,2,4-trimethyl-2H-
benzo[6,7]cyclohepta[1,2,3-de][1]benzopyran-5,10,11-triol [70] (Millard et al., 1991).
The antioxidant activities of B. tarapotensis were determined by measuring
their free radical scavenging effects using the 1,1-diphenyl-2-picryl hydrazyl free
radical (DPPH). Trolox equivalent antioxidant activity (TEAC) methods and the
coupled oxidation of β-carotene and linoleic acid. (-) Isolariciresinol-3-α-O-β-Dglucopyranoside [73], (+)-1-hydroxypinoresinol-1-O-β-D-glucopyranoside [74] and
isoacteoside [75] showed good activities in the DPPH and TEAC tests, while 2,4dihydroxy-2-(2-hydroxy ethyl) cyclohexe-5-en-1-one [71] and caffeoyl ester of
apionic acid [76] were active in the coupled oxidation of β-carotene and linoleic acid
bioassay (Braca et al., 2001).
7. Traditional Uses and Biological Activities of Croton spp.
A great number of species in the genus Croton is used in folk medicine for
wound infection and also accelerate wound healing. Moreover, they are used to treat
rheumatism, cancer (Luzbetak et al., 1979), gastric diseases (Craveiro et al., 1981a),
diarrhea, diabetes (Kubo et al., 1991), anthelmintic, purgative, skin rashes, malaria,
venereal diseases (Mazzanti et al., 1987) and whooping cough (Weckert et al., 1992).
Extracts of several species of Croton are known to produce anti-inflammatory,
antibacterial, antiviral, insecticidal, antifungal, cytotoxicity and other effects.
Detailed information on the biological activities of some Croton species is
exemplified below.
The ethyl acetate extract from bark of C. cuneatus, C. lechleri and aerial parts
of C. trinititatis showed antibacterial activity against Staphylococcus aureus and
antiviral activity against sindbis virus and murine cytomegalovirus (Macrae et al.,
1988).
The ethanol extract from bark of C. guatamalensis revealed the
antidermatomucosal infections against Candida albicans (Caceres et al., 1991). A
benzene extract of C. sonderianus was shown to have antibiotic activity against
Mycobacterium smegmatis and Staphylacoccus aureus (Craveiro and Silveira, 1982).
The extract of the cortices of C. cajucara showed anti-inflammatory activity against
62
topical inflammation in the mouse ear induced by teleocidin which was a highly
potent irritant and tumor-promoting alkaloid (Ichihara et al., 1991). The methanol
extract of the bark of C. cajucara exhibits strong insect-growth inhibitory activity in
the artificial diet feeding bioassay using the lepidopteran pest insect Pectinophora
gossypiela (pink ballworm) (Kubo et al., 1991). The ethanol extract of C. cajucara
was reported to have a lipid lowering effect in rats fed high fat diet but not in normal
rat (Farias et al., 1997). The hot petrol extract of the root of C. lacciferus and the
acetone extract of the root of C. aromaticus showed significant insecticidal activity
against Alphis craccivora (Bandara et al., 1988; Bandara et al., 1987). The extract of
the red sap from C. palanostigma was found to be cytotoxic to V-79 cells (Itokawa et
al., 1991).
An alcoholic extract of the fruits of C. macrostachys showed significant
inhibitory activity against the Lewis lung carcinoma in mice. Systematic fractionation
of the active extract led to characterization of a major active component, crotepoxide
[204], a cyclohexane diepoxide (Kupchan et al., 1969).
An acetone extract of the stem of C. sublyratus showed inhibitory activity
against reserpine–induced ulcer in mice and Shay-ulcer in rats.
Systematic
fractionation of the active acetone extract guided by antiulcer activity assay led to the
isolation of 18-hydroxy geranylgeraniol or plaunotol [173] as the principle constituent
with anti-reserpine ulcer activity. This plant known in Thai as “Plau-noi” and has
been used as anthelmintic and dermatologic agent (Ogiso et al., 1978).
The bioassay-guided fractionation of the crude extract of C. cajucara led to
the isolation of two clerodane diterpenes, cajucarinolide [85] and isocajucarinolide
[86] which showed inhibitory activities against the topical inflammation in the mouse
ear induced by teleocidin (dose = 1 µg/ear) with IC50 of 5.6 and 3.0 µg, respectively.
In addition, these compounds are potent inhibitors of bee venom phospholipase A2 in
vitro (Ichihara et al., 1991).
Taspine [190] isolated from the chloroform extract of a red viscous sap of the
bark of mature trees of C. lechleri and its hydrochloride salt were shown to have antiinflammatory activity in three different standard pharmacological models such as the
carrageenan-induced pedal edema method, the cotton pellet-induced granuloma
method and the adjuvant polyarthritis model (Perdue et al., 1979).
63
Taspine [190] isolated from C. palanostigma was found to be cytotoxic to V79 cells and KB cells with IC50 of 0.17 and 0.39 µg/ml, respectively (Itokawa et al.,
1991).
The kauranoids, ent-Kaur-15-en-3β,17-diol [132] and ent-Kaur-15β,16epoxykauran-17-ol [133] from the hot petrol extracts of the root of C. lacciferus
showed moderate insecticidal activity against Alphis craccivora (Bandara et al., 1988)
Trans-dehydrocrotonin (t-DCTN), a 19-nor clerodane diterpene [88] was
isolated from the bark of C. cajucara. This compound exhibited an insect growth
inhibitory property with ED50 of 30 ppm against the lepidopteran pest insects
(Pectinophora gossypiella and Heliothis virescens) (Kubo et al., 1991) and
demonstrated a significant hypoglycemic activity in alloxan-induced diabetic rats but
not in normal rats. The oral medication with t-DCTN (25 and 50 mg/kg) when
administered daily on three consecutive days caused a significant decrease of blood
sugar levels when compared to untreated diabetic controls (Farias et al., 1997).
8,9-Secokaurane diterpenes, ent-8,9-seco-7α,11β-Diacetoxykaura-8(14),16dien-9,15-dione [128], ent-8,9-seco-8,14-Epoxy-7α-hydroxy-11β-acetoxykaura-16kauren-9,15-dione [129] and ent-8,9-seco-7α,11β-Diacetoxykaura-8(14),16-dien9,15-dione [130] isolated from C. kongensis exhibited antimycobacterial activity with
minimum inhibitory concentrations (MICs) of 25.0, 6.25 and 6.25 µg/ml, respectively
and possessed in vitro antimalarial activity against Plasmodium falciparum (K1,
multidrug-resistant strain) (Thongtan et al., 2003)
Neocrotocembranal [151] isolated from the stem of C. oblongifolius markedly
inhibited platelet aggregation induced by thrombin (0.25 unit/ml). The effect of
neocrotocembranal on platelets is probably due to the reactive aldehyde functionality.
In addition, neocrotocembranal (6.48 µg/ml) and neocembraneic acid [150] (41.47
µg/ml) exhibited cytotoxic activity against P-388 cell culture. It should be mentioned
that many cembranoids exhibit cytotoxic activity, especially those highly
functionalized cembranoids obtained from marine sources (Roengsumran et al.,
1999b).
CHAPTER III
EXPERIMENTAL
1. Sources of Plant Materials
The stems and the roots of Bauhinia sirindhorniae K & S.S. Larsen were
collected from Nongkhai Province, Thailand in January 2001. Authentication of the
plant materials was done by comparison with herbarium specimens (BKF No.
124725) at the Botany Section, Technical Division, Department of Agriculture and
Cooperatives, Bangkok, Thailand. A voucher specimen has been deposited in the
herbarium of the Faculty of Pharmaceutical Sciences, Chulalongkorn University,
Bangkok, Thailand.
The leaves and the branches of Croton hutchinsonianus Hosseus were
collected from Karnchanaburi Province, Thailand in March 2003. Authentication of
the plant materials was done by comparison with herbarium specimens (BKF No.
2225) at the Botany Section, Technical Division, Department of Agriculture and
Cooperatives, Bangkok, Thailand. A voucher specimen has been deposited in the
herbarium of the Faculty of Pharmaceutical Sciences, Chulalongkorn University,
Bangkok, Thailand.
2. General Techniques
2.1 Analytical Thin Layer Chromatography (TLC)
Technique
:
One dimension, ascending
Adsorbent
:
1. Silica gel 60 F254 (E. Merck) precoated plate
(Aluminium sheet)
2. ODS, RP-18 F254 (E. Merck) precoated plate
(Aluminium sheet)
Layer thickness
:
0.25 mm
Distance
:
5 cm
Temperature
:
room temperature (25-35 °C)
Detection
:
1. Ultraviolet light at 254 and 365 nm
2. 10% H2SO4 in EtOH and heated at 110 °C for 10 min
65
2.2 Preparative Thin Layer Chromatography (PTLC)
Technique
:
One dimension, ascending
Adsorbent
:
Silica gel 60 F254 (E. Merck) precoated plate
Layer thickness
:
1 mm
Distance
:
15 cm
Temperature
:
room temperature (25-35 °C)
Detection
:
Ultraviolet light at 254 and 365 nm
2.3 Column chromatography
2.3.1 Vacuum Liquid Column Chromatography
Adsorbent
:
Silica gel 60 (70-230 mesh)
Packing method
:
Dry packing
Sample loading
:
The sample was dissolved in a small amount of organic
solvent, mixed with a small quantity of adsorbent,
triturated, dried and then placed gently on the top of the
column.
:
Detection
Fractions were examined by TLC observing under UV
light (254 and 356 nm).
2.3.2 Flash Column Chromatography
:
Adsorbent
1. Silica gel 60 (230-400 mesh)
2. Cosmosil 75 C18-OPN (Nacalai tesque)
Packing method
:
Dry packing
Sample loading
:
The sample was dissolved in a small amount of eluent
and then applied gently on the top of the column.
:
Detection
Fractions were examined in the same manner as
described in section 2.3.1
2.3.3 Gel Filtration Chromatograaphy
Gel filter
:
Sephadex LH 20 (Pharmacia)
Packing method
:
Gel filter was suspended in the eluent and left standing
to swell for 24 hours prior to use. It was then poured
into the column and allowed to set tightly.
Sample loading
:
The sample was dissolved in a small volume of eluent
and applied on top of the column.
66
2.3.4 High Pressure Liquid Chromatography (HPLC)
Column (Semi-prep.):
(Analytical)
Flow rate
Inertsil ODS column (20 i.d.×250mm) (gaskurokogyo)
:
TSK gel ODS120A (4.6 i.d.×150 mm) (TOSOH)
:
1. 5 ml/min for semi-preparative column
2. 1 ml/min for analytical column
Mobile phase
:
1. Isocratic 85% water + 25% methanol
2. Isocratic 70% water + 20% acetonitrile + 10%
methanol
Sample preparation :
The sample was dissolved in a small amount of eluent
and filtered through Millipore filter paper before
injection.
Injection volume
:
1 ml
Pump
:
LC-9A (Shimadzu)
Detector
:
SPD-6AV UV Detector (Shimadzu)
Recorder
:
C-R6A Chromatopac (Shimadzu)
Temperature
:
Room temperature
2.4 Spectroscopy
2.4.1 Ultraviolet (UV) Absorption Spectra
UV spectra were obtained on Shimadzu UV-2100S UV/vis spectrophotometer
(Chulabhorn Research Institute).
2.4.2 Infrared (IR) Absorption spectra
IR spectra were recorded on a JASCO A-302 (Chulabhorn Research Institute)
and a JAS FT/IR 230-IR spectrometer (Faculty of Pharmaceutical Sciences, Chiba
University).
2.4.3 Mass Spectra
Fast-Atom Bombardment mass spectra (FABMS) and High Resolution Fast
Atom Bombardment mass spectra (HRFABMS) were measured on a JEOL JMS-HX110A spectrometer (The Chemical Analysis Center, Chiba University).
Electron impact mass spectra (EIMS) were measured on a Finnigan INCOS 50
and High Resolution Fast Atom Bombardment mass spectra (HRFABMS) were
measured on a MAT 90 (Chulabhorn Research Institute).
67
2.4.4 Proton and Carbon-13 Nuclear Magnetic Resonance (1H and
13
C-
NMR) Spectra
1
H NMR (400 MHz),
13
C NMR (100 MHz) spectra were obtained with a
Bruker AM 400 (Chulabhorn Research Institute).
1
H NMR (500 MHz),
13
C NMR (125 MHz) spectra were obtained with a
JEOL JNM GSX 500A spectrometer (Faculty of Pharmaceutical Sciences, Chiba
University).
2.5 Physical Properties
2.5.1 Optical Rotations
Optical rotation were measured on a JASCO DIP 140 polarimeter (Faculty of
Pharmaceutical Sciences, Chiba University) and a Perkin Elmer 341 polarimeter
(Pharmaceutical Research Instrument Center, Faculty of Pharmaceutical Sciences,
Chulalongkorn University).
2.5.2 Circular Dichroism (CD) Spectra
Circular Dichroism spectra were measured on a JASCO CD J-720 W
spectrometer (Faculty of Pharmaceutical Sciences, Chiba University).
2.5.3 Melting Points
Melting points were obtained on a Yanagimoto Micro Melting Point
Apparatus (Faculty of Pharmaceutical Sciences, Chiba University) and Eletrothermal
melting point apparatus, Electrothermal 9100 (Chulabhorn Research Institite).
2.6 Solvents
Throughout this work, commercial grade organic solvents were used and
redistilled prior to use.
2.7 Chemicals
1,1-Diphenyl-2-picrylhydrazyl (DPPH) (Wako)
6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (Trolox) (Aldrich)
Quercetin (Aldrich)
2.8 Microtiter Plate Reader
Microtiterplate reader was performed on a Biorad Model 550 (Faculty of
Pharmaceutical Sciences, Chiba University).
68
3. Extraction and Separation
3.1 Extraction and Separation of the Stems of Bauhinia sirindhorniae
3.1.1 Extraction
The dried stems of Bauhinia sirindhorniae (350 g) were successively extracted
with hexane (3×4 L), chloroform (3×4 L), and 95% ethanol (3×4 L). The filtrates
were pooled and evaporated under reduced pressure at the temperature not exceeding
40 °C to give the corresponding hexane (620.5 mg), chloroform (515.3 mg) and 95%
ethanol extract (62.3 g), respectively.
The 95% ethanol extract (62.3 g) was then partitioned between butanol and
water. The butanol layer was dried to yield 22.5 g of a butanol extract while 20.4 g of
an aqueous extract was obtained.
3.1.2 Isolation
3.1.2.1 Isolation of Compounds from Chloroform Extract (Sheme 3 and
Figure 3)
The chloroform extract (515.3 mg) was dissolved in a small amount of
chloroform, triturated with silica gel 60 (70-230 mesh) and dried at room temperature.
It was then fractionated by liquid column chromatography. Elution was completed in
a polarity gradient manner with mixtures of chloroform-acetone.
The eluates were examined by TLC (silica gel) using mixtures of chloroformacetone as a developing solvent. Fractions with similar chromatographic pattern were
combined to afford five fractions: fractions SC-A (27.6 mg), SC-B (100.2 mg), SC-C
(68.9 mg), SC-D (91.8 mg) and SC-E (22.1 mg).
3.1.2.1.1 Isolation of Compound BSC1 (Lupeol)
Fraction SC-B (100.2 mg) was separated by column chromatography (silica
gel 60 (230-400 mesh)) using mixtures of hexane-acetone (9:1) as eluent. After
combining of collected fractions according to chromatographic pattern (silica gel,
hexane-acetone 4:1), three fractions including SC-B1 to SC-B3 were obtained.
Fraction SC-B1 (23.4 mg), after removal of solvents, gave compound BSC1
(5.7 mg, 1.6×10-3% based on dried weight of stems). This compound was identified
as lupeol [77].
3.1.2.1.2 Isolation of Compound BSC2 (Glutinol)
Fraction SC-B2 (52.3 mg) was subjected to gel filtration chromatography
using a Sephadex LH 20 column with mixtures of chloroform-acetone (4:1) as the
69
eluent.
The eluates were collected and combined according to their TLC
chromatographic patterns (silica gel, hexane-acetone 4:1).
Fraction SC-B2-2 (35.5 mg), after removal of solvents, gave compound BSC2
(16.3 mg, 4.7×10-3% based on dried weight of stems). This compound was identified
as glutinol [214].
3.1.2.2 Isolation of Compounds from Butanol Extract (Schemes 4-5
and Figure 3)
The butanol extract (22.5 g) was dissolved in a small amount of methanol,
triturated with silica gel 60 (70-230 mesh) and dried at room temperature. It was then
fractionated by liquid column chromatography. Elution was completed in a polarity
gradient manner with mixtures of chloroform-methanol-water.
The eluates were examined by TLC (silica gel) using mixtures of chloroformmethanol-water as a developing solvent. Fractions with similar chromatographic
pattern were combined to afford seven fractions: fractions SB-A (51.2 mg), SB-B
(81.4 mg), SB-C (810.6 mg), SB-D (1.2 g), SB-E (393.8 mg), SB-F (2.1 g) and SB-G
(16.5 g).
3.1.2.2.1 Isolation of Compound BSB1 (Isoliquiritigenin)
Fraction SB-A (51.2 mg) was fractionated by gel filtration chromatography
using a Sephadex LH 20 column with mixtures of chloroform-acetone (1:1) as an
eluent. Eluates were collected and combined based on their chromatographic patterns
(silica gel, hexane-ethyl acetate 3:2) to give four fractions (SB-A1 to SB-A4).
Fraction SB-A3 (8.2 mg), after removal of solvents, gave compound BSB1
(4.5 mg, 1.3×10-3% based on dried weight of stems). This compound was identified
as isoliquiritigenin [14].
3.1.2.2.2 Isolation of Compound BSB2 ((+)-Isolariciresinol-3α-O-α-Lrhamnoside)
Fraction SB-C (810.6 mg) was separated on a Sephadex LH 20 column
(chloroform-methanol 1:1). The eluates were collected and examined by TLC (silica
gel, chloroform-methanol-water 8:2:0.1).
Fractions with similar chromatographic
patterns were combined to yield four fractions (fraction SB-C1 to SB-C4).
Fraction SB-C2 (100.5 mg) was re-separated by column chromatography
using silica gel 60 (230-400 mesh) as an adsorbent and eluted with mixtures of
chloroform-methanol-water (8:2:0.1).
The eluates were collected and combined
70
according to similarity of chromatographic patterns (chloroform-methanol-water
8:2:0.5) to obtained four fractions (SB-C2-1 to SB-C2-4).
Fraction SB-C2-3 (28.9 mg) was subsequently separated by HPLC using an
Inertsil ODS column (20 i.d.×250 mm) with UV 254 nm detection. Elution was
performed in an isocratic manner with 70% water + 20% acetonitrile + 10% methanol
(flow rate 5 ml/min). Compound BSB2 (2.0 mg, 5.7×10-4% based on dried weight of
stems) was obtained at the retention time of 17 minutes.
This compound was
identified as (+)-isolariciresinol 3α-O-α-L- rhamnoside [215].
3.1.2.2.3 Isolation of Compound BSB3 (3,4,5-Trimethoxyphenolic-1-Oβ-D- glucoside)
Fraction SB-C3 (112.8 mg) was re-separated by column chromatography
using silica gel 60 (230-400 mesh) as an adsorbent and eluted with mixtures of
chloroform-methanol-water (8:2:0.1).
The eluates were collected and combined
according to the chromatographic patterns (silica gel, chloroform-methanol-water
8:2:0.5) to give four fractions (SB-C3-1 to SB-C3-4).
Fraction SB-C3-3 (10.6 mg) was separated by HPLC using an Inertsil ODS
column (20 i.d.×250 mm) eluted with in an isocratic manner with 70% water + 20%
acetonitrile + 10% methanol (flow rate 5 ml/min) to give compound BSB3 (3.8 mg,
1.1×10-3% based on dried weight of stems) at retention time 21 minutes. It was
identified as 3,4,5-trimethoxyphenolic-1-O-β-D- glucoside [216].
3.1.2.2.4 Isolation of Compound BSB4 ((-)-Epicatechin)
Fraction SB-C4 (150.3 mg) was subjected to column chromatography using
silica gel 60 (230-400 mesh) as an adsorbent and eluted with mixtures of chloroformmethanol (9:1).
After combination of collected fractions according to
chromatographic pattern (silica gel, chloroform-methanol 4:1), four fractions
including fraction SB-C4-1 to SB-C4-4 were obtained.
Recrystallization of fraction SB-C4-2 (22.7 mg) with mixtures of chloroformmethanol yielded a pale yellow needle of compound BRB4 (8.5 mg, 2.4×10-3% based
on dried weight of stems). This compound was identified as (-)-epicatechin [217].
3.1.2.2.5 Isolation of Compound BSB5 (Protocatechuic acid)
Fraction SB-D (1.2 g) was further purified by gel filtration chromatography
using a Sephadex LH 20 column with mixtures of chloroform-methanol (1:1) as the
eluent, which resulted in the collection of two fractions SB-D1 and SB-D2.
71
Fraction SB-D-2 (564.4 mg) was subjected to Cosmosil 75 C18-OPN column
chromatography with mixtures of methanol-water (1:4). Eluates were collected and
combined based on their chromatographic patterns (silica gel, chloroform-methanol
4:1) to give four fractions (SB-D2-1 to SB-D2-4). Fraction SB-D2-2 (22.6 mg) was
recrystallized from methanol to give compound BSB5 (8.0 mg, 2.3×10-3% based on
dried weight of stems) as a colorless needle. This compound was identified as
protocatechuic acid [218].
3.1.2.2.6 Isolation of Compound BSB6 (Lithospermoside)
Fraction SB-E (393.8 mg) was separated by a Cosmosil 75 C18-OPN column
chromatography. Elution was performed in a polarity isocratic manner with the
mixtures of methanol-water (1: 9). Eluates with similar TLC behavior (silica gel,
chloroform-methanol-water 7:3:1) were pooled to give four fractions (SB-E1 to SBE4).
Fraction SB-E2 (30.2 mg) was re-separated by a Cosmosil 75 C18-OPN
column chromatography using and eluted with mixtures of methanol-water (1:9). The
eluates were collected and combined according to the chromatographic patterns (silica
gel, chloroform-methanol-water 8:2:0.1) to obtain three fractions (SB-E2-1 to SB-E23). Fraction SB-E2-1 (12.4 mg), after removal of solvents, gave compound BSB6
(5.5 mg, 1.6×10-3% based on dried weight of stems). This compound was identified
as lithospermoside [54].
3.2 Extraction and Separation of the Roots of Bauhinia sirindhorniae
3.2.1 Extraction
The dried roots of Bauhinia sirindhorniae (300 g) were successively extracted
with hexane (3×3 L), chloroform (3×3 L), and 95% ethanol (3×3 L). The filtrates
were pooled and evaporated under a reduced pressure at the temperature not
exceeding 40 °C to give the corresponding hexane (520.6 mg), chloroform (490.5 mg)
and 95% ethanol extract (58.2 g), respectively.
The ethanol extract (58.2 g) was then partitioned between butanol and water.
The butanol layer was dried to yield 22.3 g of a butanol extract while 19.5 g of an
aqueous extract was obtained.
3.2.2 Isolation
3.2.2.1 Isolation of Compounds from Chloroform Extract (Scheme 6 and
Figure 4)
72
The chloroform extract (490.5 mg) was dissolved in a small amount of
chloroform, triturated with silica gel 60 (70-230 mesh) and dried at room temperature.
It was then fractionated by liquid column chromatography. Elution was completed in
a polarity gradient manner with mixtures of chloroform-methanol.
The eluates were examined by TLC using chloroform-methanol as a
developing solvent. Fractions with similar chromatographic pattern were combined to
afford five fractions: fractions RC-A (62.1 mg), RC-B (121.9 mg), RC-C (157.3 mg),
RC-D (32.0 mg) and RC-E (86.0 mg).
3.2.2.1.1
Isolation of Compound BRC1 (5,7-Dihydroxychromone)
Fraction RC-B (121.9 mg) was separated on a Sephadex LH 20 column
(chloroform-methanol 1:1). Eluates were collected and combined based on their
chromatographic patterns (silica gel, chloroform-ethyl acetate 98:2) to give four
fractions (RC-B1 to RC-B4).
Fraction RC-B2 (14.6 mg) was recrystallized from a mixture of chloroformmethanol to give compound BRC1 (7.2 mg, 2.4×10-3% based on dried weight of
roots) as a colorless needle. This compound was identified as 5,7 dihydroxychromone
[219].
3.2.2.1.2
Isolation of Compound BRC2 (Sitosteryl-3-O-β-D-glucoside)
Fraction RC-D (32.0 mg) was fractionated by gel filtration chromatography
using a Sephadex LH 20 column with mixtures of chloroform-methanol (1:1) as the
eluent. Compound BRC2 (10.3 mg, 3.4×10-3% based on dried weight of roots) was
finally obtained after the removal of solvent from fraction RC-D2 (19.2 mg). This
compound was later identified as sitosteryl-3-O-β-D-glucoside [37].
3.2.2.3 Isolation of Compounds from Butanol Extract (Schemes 7-8 and
Figure 4)
The butanol extract (22.3 g) was dissolved in a small amount of methanol,
triturated with silica gel 60 (70-230 mesh) and dried at room temperature. It was then
fractionated by liquid column chromatography. Elution was completed in a polarity
gradient manner with mixtures of chloroform-methanol-water.
The eluates were examined by TLC using mixtures of chloroform-methanol as
a developing solvent. Fractions with similar chromatographic pattern were combined
to afford six fractions: fractions RB-A (285.0 mg), RB-B (120.2 mg), RB-C (329.3
mg), RB-D (790.2 mg), RB-E (2.5 g) and RB-F (10.2 g).
73
3.2.2.3.1 Isolation of Compound BRB1 ((2S)-Naringenin)
Fraction RB-A (285.0 mg) was separated on silica gel 60 (230-400 mesh) as
an adsorbent.
Elution was performed in an isocratic manner with mixtures of
chloroform-methanol (98:2).
Eluates with similar TLC behavior (silica gel,
chloroform-methanol 98:2) were pooled to give four fractions (RB-A1 to RB-A4).
Fraction RB-A2 (52.5 mg) was recrystallized from a mixture of chloroformmethanol to give compound BRB1 (3.7 mg, 1.2×10-3% based on dried weight of
roots). It was identified as (2S)-naringenin [17].
3.2.2.3.2 Isolation of Compound BRB2 (Luteolin)
Fraction RB-B (120.2 mg) was subjected to column chromatography using
silica gel 60 (230-400 mesh) as an adsorbent and eluted with mixtures of chloroformmethanol (95:5).
After combination of collected fractions according to
chromatographic pattern (silica gel, chloroform-methanol 9:1), four fractions
including fraction RB-B1 to RB-B4 were obtained.
Fraction RB-B3 (48.0 mg) was further fractionated by gel filtration
chromatography using a Sephadex LH 20 column with a mixture of chloroformmethanol (1:1) as the eluent. Fraction RB-B3-2 (24.0 mg) was recrystallization from
a mixture of chloroform and methanol to give compound BRB2 (3.0 mg, 1.0×10-3%
based on dried weight of roots) as a yellow needle. It was identified as luteolin [220].
3.2.2.3.3 Isolation of Compound BRB3 ((2S)-Eriodictyol)
Fraction RB-C (329.3 mg) was further purified by repeated column
chromatography using silica gel 60 (230-400 mesh) as an adsorbent and eluted with
mixtures of chloroform-methanol (9:1), which resulted in the collecting of fractions
RB-C1 to RB-C3.
Fraction RB-C1 (43.3 mg) was subjected to gel filtration chromatography
using a Sephadex LH 20 column with a mixture of chloroform-methanol (1:1) as the
an eluent.
Recrystallization of fraction RB-C1-2 (24.6 mg) with a mixture of
chloroform and methanol yielded a pale yellow needle of compound BRB3 (7.3 mg,
2.4×10-3% based on dried weight of roots). This compound was identified as (2S)eriodictyol [16].
3.2.2.3.4 Isolation of Compound BRB4 ((+)-Taxifolin)
Fraction RB-C3 (97.2 mg) was subjected to column chromatography using
silica gel 60 (230-400 mesh) as an adsorbent. Elution with mixtures of chloroform-
74
methanol (9:1).
After combination of collected fractions based on their
chromatographic behavior (silica gel, chloroform-methanol 9:1) leading to three
fractions: fractions RB-C3-1 to RB-C3-3 were obtained.
Recrystallization of fraction RB-C3-2 (32.1 mg) with mixtures of chloroform
and methanol yielded a pale yellow needle of compound BRB4 (8.9 mg, 3.0×10-3%
based on dried weight of roots). This compound was identified as (+)-taxifolin [221].
3.2.2.3.5 Isolation of Compound BRB5 ((+) Lyoniresinol-3α-O-α-Lrhamnoside)
Fraction RB-D (790.2 mg) was fractionated by column chromatography using
silica gel 60 (230-400 mesh) as an adsorbent and eluted with mixtures of chloroformmethanol-water (8:2:0.1). The eluates were collected and combined according to the
chromatographic patterns (silica gel, chloroform-methanol-water 7:2:1) to obtained
four fractions (RB-D1 to RB-D4).
Purification of fraction RB-D1 (166.9 mg) was further performed by gel
filtration chromatography using a Sephadex LH 20 column with mixtures of
chloroform: methanol (1:1) as an eluent. The eluates were collected and combined
based on their TLC chromatographic patterns (silica gel, chloroform-methanol-water
7:3:1) to give two fractions (RB-D1-1 and RB-D1-2).
Fraction RB-D1-2 was
fractionated by column chromatography (Cosmosil 75C18-OPN, methanol: water 1:4)
to give a colorless amorphours mass of compound BRB5 (30.0 mg, 1.0×10-2% based
on dried weight of roots). This compound was identified as (+) lyoniresinol-3α-O-αL-rhamnoside [222].
3.2.2.3.6 Isolation of Compound BRB6 (5-Hydroxychromone-7-β-Dglucoside)
Fraction RB-D2 (78.5 mg) was further fractionated by gel filtration
chromatography using a Sephadex LH 20 column with mixtures of chloroformmethanol (1:1) as an eluent. The eluates were collected and combined based on their
TLC chromatographic patterns (silica gel, chloroform-methanol-water 8:2:0.1) to give
two fractions (RB-D2-1 and RB-D2-4).
Fraction RB-D2-3 (8.9 mg) was recrystallized from a mixture of chloroform
and methanol to give compound BRB6 (1.0 mg, 3.0×10-4 % based on dried weight of
roots) as a yellow needle. This compound was identified as 5-hydroxychromone-7-βD-glucoside [223].
75
3.2.2.3.7 Isolation of Compound BRB7 (Menisdaurin)
Fraction RB-D3 (68.2 mg) was fractionated by gel filtration chromatography
using a column of a Sephadex LH 20 with mixtures of chloroform: methanol (1:1) as
an eluent.
The eluates were collected and combined based on their TLC
chromatographic patterns (silica gel, chloroform-methanol-water 7:3:1) to give two
fractions (RB-D3-1 and RB-D3-2).
Fraction RB-D3-2 (63.7 mg) was purified by HPLC using an Inertsil ODS
column (20 i.d.×250 mm) eluted with in an isocratic manner with UV 254 nm
detection. Elution was performed in an isocratic manner with 85% water + 25%
methanol (flow rate 5 ml/min. A total of compound BRB7 (3.2 mg, 1.1×10-3% based
on dried weight of roots) was obtained at the retention time of 25 minutes. This
compound was subsequently identified as menisdaurin [224].
3.3 Extraction and Separation of the Leaves of Croton hutchinsonianus
3.3.1 Extraction
The dried leaves of (2.5 kg) Croton hutchinsonianus were successively
extracted with hexane (3×20 L), ethyl acetate (3×20 L), and 95% ethanol (3×20 L).
The filtrates were pooled and evaporated under reduced pressure at the temperature
not exceeding 40 °C to give the corresponding hexane (106.8 g), ethyl acetate (110.7
g) and 95% ethanol extract (112.4 g), respectively.
The 95% ethanol extract (112.4 g) was then partitioned between butanol and
water. The butanol layer was dried to yield 75.5 g of a butanol extract whereas 18.4 g
of an aqueous extract was obtained.
3.3.2 Isolation
3.3.2.1 Isolation of Compounds from Ethyl Acetate Extract (Scheme 9
and Figure 5)
The ethyl acetate extract (110.7 g) was dissolved in a small amount of
chloroform, triturated with silica gel 60 (70-230 mesh) and dried at room temperature.
It was then fractionated by vacuum liquid column chromatography. Elution was
completed in a polarity gradient manner with mixtures of hexane-ethyl acetate.
The eluates were examined by TLC (silica gel) using hexane-ethyl acetate as a
developing solvent. Fractions with a similar chromatographic pattern were combined
to afford four fractions: fractions LE-A (4.3 g), LE-B (20.2 g), LE-C (48.6 g) and
LE-D (25.8 g).
76
3.3.2.1.1 Isolation of Compound CBE1 (Farnesyl acetone)
Fraction LE-A (4.3 g) was separated on silica gel 60 (230-400 mesh) as an
adsorbent. Elution was performed in an isocratic manner with mixtures of hexaneethyl acetate (98:2). Eluates with a similar TLC behavior (silica gel, hexane-ethyl
acetate 95:5) were pooled to give three fractions (LE-A1 to LE-A3).
Fraction LE-A2 (33.5 mg) was separated by preparative TLC using hexane
and ethyl acetate as a developing solvent to give compound CBE1 (19.8 mg,
7.9×10-4% based on dried weight of leaves). It was identified as a farnesyl acetone
[225].
3.3.2.1.2 Isolation of Compound CBE2 (Poilaneic acid)
Fraction LE-B (20.3 g) was fractionated by gel filtration chromatography
using a Sephadex LH 20 column with a mixture of dichloromethane-acetone (1:1) as
the eluent.
The eluates were collected and combined based on their TLC
chromatographic patterns (silica gel, hexane-ethyl acetate 4:1) to give two fractions
(LE-B1 and LE-B2).
Fraction LE-B2 (10.1 mg) was re-separated on column chromatography using
a Cosmosil 75C18-OPN column and eluted with mixtures of methanol-water (9:1).
The eluates were collected and combined according to similarity of chromatographic
patterns (silica gel, hexane-ethyl acetate 4:1) to obtained three fractions (LE-B2-1 to
LE-B2-3). Fraction LE-B2-2 (98.9 mg), after removal of solvent gave compound
CBE2 (48.8 mg, 2.0×10-3% based on dried weight of leaves). This compound was
identified as poilaneic acid [226].
3.3.2.1.3 Isolation of Compound CBE4 (3-(4-Hydroxy-3,5-dimethoxy
phenyl)-propyl benzoate)
Fraction LE-C (48.6 g) was fractionated by gel filtration chromatography
using a Sephadex LH 20 column with mixtures of dichloromethane-acetone (1:1) as
the eluent.
The eluates were collected and combined based on their TLC
chromatographic patterns (silica gel, hexane-ethyl acetate 4:1) to give two fractions
(LE-C1 and LE-C2).
Fraction LE-C2 (5.8 g) was separated by preparative TLC using hexane-ethyl
acetate as a developing solvent to give compound CBE4 (98.5 mg, 3.9×10-3% based
on dried weight of leaves). It was identified as 3-(4-hydroxy-3,5-dimethoxyphenyl)propyl benzoate [227].
77
3.4 Extraction and Separation of the Branches of Croton hutchinsonianus
3.4.1 Extraction
The dried branches of (1.3 kg) Croton hutchinsonianus were successively
extracted with hexane (3×10 L), ethyl acetate (3×10 L), and 95% ethanol (3×10 L).
The filtrates were pooled and evaporated under reduced pressure at the temperature
not exceeding 40 °C to give the corresponding hexane (15.0 g), ethyl acetate (20.7 g)
and 95% ethanol extract (27.0 g), respectively.
The 95% ethanol extract (27.0 g) was then partitioned between butanol and
water. The butanol layer was dried to yield 12.5 g of a butanol extract while 10.4 g of
an aqueous extract was obtained.
3.4.2 Isolation
3.4.2.1 Isolation of Compounds from Ethyl Acetate Extract (Schemes 10
and Figure 5)
The ethyl acetate extract (20.7 g) was dissolved in a small amount of
chloroform, triturated with silica gel 60 (70-230 mesh) and dried at the room
temperature. It was then fractionated by vacuum liquid column chromatography.
Elution was completed in a polarity gradient manner with mixtures of hexane-ethyl
acetate.
The eluates were examined by TLC (silica gel) using hexane-ethyl acetate as a
developing solvent. Fractions with similar chromatographic pattern were combined to
afford four fractions: fractions BE-A (1.2 g), BE-B (3.5 g), BE-C (5.7 g) and BE-D
(6.9 g).
3.4.2.1.1 Isolation of Compound CBE1 (Farnesyl acetone)
Fraction BE-A (1.2 g) was separated on silica gel 60 (230-400 mesh) as an
adsorbent. Elution was performed in a polarity isocratic manner with mixtures of
hexane-ethyl acetate (98:2). Eluates with similar TLC behavior (silica gel, hexaneethyl acetate 95:5) were pooled to give three fractions (BE-A1 to BE-A3).
Fraction BE-A2 (21.4 mg) was separated by preparative TLC using hexane
and ethyl acetate as a developing solvent to give compound CBE1 (2.1 mg, 1.8×10-4%
based on dried weight of branches). It was identified as farnesyl acetone [225].
3.4.2.1.2 Isolation of Compound CBE2 (Poilaneic acid)
Fraction BE-B (3.5 g) was fractionated by gel filtration chromatography using
a Sephadex LH 20 column with mixtures of dichloromethane-acetone (1:1) as the
78
eluent.
The eluates were collected and combined based on their TLC
chromatographic patterns (silica gel, hexane-ethyl acetate 4:1) to give two fractions
(BE-B1 and BE-B2).
Fraction BE-B2 (1.1 g) was re-separated on column chromatography using a
Cosmosil 75 C18-OPN column and eluted with the mixtures of methanol-water (9:1).
The eluates were collected and combined according to similarity of chromatographic
patterns (silica gel, hexane-ethyl acetate 4:1) to obtained three fractions (BE-B2-1 to
BE-B2-3). Fraction BE-B2-2 (25.6 mg), after removal of solvents, gave compound
CBE2 (5.7 mg, 4.8×10-4% based on dried weight of branches). This compound was
identified as poilaneic acid [163].
3.4.2.1.3 Isolation of Compound CBE3 (4-Hydroxybenzaldehyde)
Fraction BE-C (5.7 g) was fractionated by gel filtration chromatography using
a Sephadex LH 20 column with mixtures of dichloromethane-acetone (1:1) as the
eluent.
The eluates were collected and combined based on their TLC
chromatographic patterns (silica gel, hexane-ethyl acetate 4:1) to give two fractions
(BE-C1 and BE-C2).
Fraction BE-C1 (3.4 g) was recrystallized to give compound CBE3 (11.6 mg,
9.7×10-4% based on dried weight of branches) from mixtures of hexane-ethyl acetate
as a colorless needle.
This compound was identified as 4-hydroxybenzaldehyde
[226].
3.4.2.1.4 Isolation of Compound CBE4 (3-(4-Hydroxy-3,5-dimethoxy
phenyl)-propyl benzoate)
Fraction BE-C2 (1.3 g) was separated on silica gel 60 (230-400 mesh) as an
adsorbent. Elution was performed in a polarity isocratic manner with mixtures of
hexane-ethyl acetate (9:1). Eluates with similar TLC behavior (silica gel, hexaneethyl acetate 4:1) were pooled to give three fractions (BE-C2-1 to BE-C2-3).
Fraction BE-C2-2 (27.2 mg) was separated by preparative TLC using the
mixtures of hexane-ethyl acetate (3:2) as a developing solvent to give compound
CBE4 (35.6 mg, 3.0×10-3% based on dried weight of branches). It was identified as
3-(4-hydroxy-3,5-dimethoxyphenyl)-propyl benzoate [227].
3.4.2.1.5 Isolation of Compound CBE5 (Dihydroconiferyl benzoate)
Fraction BE-C2-1 (27.2 mg) was separated by preparative TLC using mixtures
of hexane-ethyl acetate (4:1) as a developing solvent to give compound CBE5 (18.9
79
mg, 1.6×10-3% based on dried weight of branches).
It was identified as
dihydroconiferyl benzoate [228].
3.4.2.1.6 Isolation of Compound CBE6 (3-(4-Hydroxyphenyl)-propyl
benzoate)
Fraction BE-C2-3 (11.5 mg) was separated by preparative TLC using mixtures
of hexane and ethyl acetate (3:2) as a developing solvent to give compound CBE6
(4.9 mg, 4.1×10-4% based on dried weight of branches). It was identified as 3-(4hydroxyphenyl)-propyl benzoate [229].
80
CHCl3 Extract (515.3 mg) from stems of Bauhinia sirindhorniae
Liquid column chromatography
Silica gel, chloroform: acetone
Fr. SC-A
(27.6 mg)
Fr. SC-C
(68.9 mg)
Fr. SC-B
(100.2 mg)
Fr. SC-D
(91.8 mg)
Silica gel
Hexane: acetone
(9:1)
Fr. SC-B1
(23.4 mg)
Fr. SC-B3
(15.9 mg)
Fr. SC-B2
(52.3 mg)
Sephadex LH 20
CHCl3: acetone
(4:1)
BSC1 (5.7 mg)
Fr. SC-B2-1
(11.2 mg)
Fr. SC-B2-2
(35.5 mg)
BSC2 (16.3 mg)
Scheme 3 Separation of the CHCl3 extract of the stems of Bauhinia sirindhorniae
Fr. SC-E
(22.1 mg)
81
Butanol Extract (22.5 g) from stems of Bauhinia sirindhorniae
Liquid column chromatography
Silica gel, chloroform: methanol: water
Fr. SB-A
(51.2 mg)
Fr. SB-B
(81.4 mg)
Fr. SB-C
(810.6 mg)
Fr. SB-D
(1.2 g)
Sephadex LH 20
CHCl3: acetone
(1:1)
Fr. SB-A1
(14.3 mg)
Sephadex LH 20
CHCl3: methanol
(1:1)
Fr. SB-A2 Fr. SB-A3
(10.4 mg) (8.2 mg)
Fr. SB-A4
(15.9 mg)
Fr. SB-D1
(485.2 mg)
Fr. SB-D2
(564.4 mg)
Fr. SB-E1 Fr. SB-E2
(22.0 mg) (30.2 mg)
Fr. SB-E3
(122.3 mg)
Fr. SB-E4
(119.5 mg)
Cosmosil
MeOH: H2O
(1:9)
Fr. SB-D2-1 Fr. SB-D2-2 Fr. SB-D2-3 Fr. SB-D2-4 Fr. SB-E2-1
(22.6 mg)
(69.8 mg)
(145.5 mg)
(210.0 mg)
(12.4 mg)
Fr. SB-E2-2
(7.1 mg)
Fr. SB-E2-3
(7.9 mg)
Recrystallization
BSB5 (8.0 mg)
Fr. SB-G
(16.5 g)
Cosmosil
MeOH: H2O
(1:9)
Cosmosil
MeOH: H2O
(1:4)
BSB1 (4.5 mg)
Fr. SB-F
(2.1 g)
Fr. SB-E
(393.8 mg)
BSB6 (5.5 mg)
Scheme 4 Separation of the butanol extract of the stems of Bauhinia sirindhorniae
82
Fr. SB-C (810.6 mg)
Sephadex LH 20
CHCl3: methanol
(1:1)
Fr. SB-C1
(287.9 mg)
Fr. SB-C2
(100.5 mg)
Fr. SB-C3
(112.8 mg)
Silica gel
CHCl3: MeOH: H2O
(8:2:0.1)
Fr. SB-C2-1
(33.3 mg)
Fr. SB-C2-2
(25.5 mg)
Fr. SB-C2-3 Fr. SB-C2-4
(17.2 mg)
(28.9 mg)
Fr. SB-C4
(150.3 mg)
Silica gel
CHCl3: MeOH: H2O
(8:2:0.1)
Fr. SB-C3-1
(13.6 mg)
Fr. SB-C3-2
(22.7 mg)
Fr. SB-C3-3
(10.6 mg)
Fr. SB-C3-4
(45.8 mg)
Silica gel
CHCl3: MeOH
(9:1)
Fr. SB-C4-1
(34.2 mg)
Fr. SB-C4-2
Fr. SB-C4-3
(22.7 mg)
(10.6 mg)
Recrystallization
ODS HPLC
MeOH: CH3CN: H2O
(1:2:7)
ODS HPLC
MeOH: CH3CN: H2O
(1:2:7)
BSB4 (8.5 mg)
BSB2 (2.0 mg)
BSB3 (3.8 mg)
Scheme 5 Separation of fraction SB-C from the butanol extract of the stems of Bauhinia sirindhorniae
Fr. SB-C-4-4
(75.1 mg)
83
CHCl3 Extract (490.5 mg) from roots of Bauhinia sirindhorniae
Liquid column chromatography
Silica gel, chloroform: methanol
Fr. RC-A
(62.1 mg)
Fr. RC-C
(157.3 mg)
Fr. RC-B
(121.9 mg)
Sephadex LH 20
CHCl3: MeOH
(1:1)
Fr. RC-B1
(35.2 mg)
Fr. RC-B2
(14.6 mg)
Fr. RC-B3
(27.2 mg)
Fr. RC-B4
(19.2 mg)
Fr. RC-E
(86.0 mg)
Fr. RC-D
(32.0 mg)
Sephadex LH 20
CHCl3: MeOH
(1:1)
Fr. RC-D1
(10.6 mg)
Fr. RC-D2
(19.2 mg)
Recrystallization
BRC1 (7.2 mg)
BRC2 (10.3 mg)
Scheme 6 Separation of the CHCl3 extract of the roots of Bauhinia sirindhorniae
84
Butanol Extract (22.3 g) from roots of Bauhinia sirindhorniae
Liquid column chromatography
Silica gel, chloroform: methanol: water
Fr. RB-A
(285.0 mg)
Fr. RB-B
(120.2 mg)
Silica gel
CHCl3: MeOH
(98:2)
Fr. RB-B1
(11.2 mg)
Fr. RB-A1
(22.8 mg)
Fr. RB-C
(329.3 mg)
Silica gel
CHCl3: MeOH
(95:5)
Fr. RB-B2
(18.4 mg)
Fr. RB-A2 Fr. RB-A3
(52.5 mg) (42.2 mg)
Sephadex LH 20
CHCl3: MeOH
(1:1)
Recrystallization
Fr. RB-B3-1
(16.0 mg)
BRB1 (3.7 mg)
Fr. RB-B3-2
(24.0 mg)
Fr. RB-C1 Fr. RB-C2
(43.3 mg) (26.5 mg)
Fr. RB-C3
(97.2 mg)
Fr. RB-C1-1
(15.1 mg)
Fr. RB-C1-2
(24.6 mg)
Fr. RB-D1
(166.9 mg)
Fr. RB-C3-1
(28.2 mg)
Fr. RB-C3-2 Fr. RB-C3-3
(32.1 mg)
(19.0 mg)
Recrystallization
Recrystallization
Recrystallization
BRB3 (7.3 mg)
Fr. RB-F
(10.2 g)
Fr. RB-D2 Fr. RB-D3 Fr. RB-D4
(78.5 mg) (168.2 mg) (196.3 mg)
Silica gel
CHCl3: MeOH
(9:1)
Sephadex LH 20
CHCl3: MeOH
(1:1)
Fr. RB-E
(2.5 g)
Silica gel
CHCl3: MeOH: H2O
(8:2:0.1)
Silica gel
CHCl3: MeOH
(9:1)
Fr. RB-B3 Fr. RB-B4
(48.0 mg) (21.6 mg)
Fr. RB-A4
(39.6 mg)
Fr. RB-D
(790.2 mg)
BRB4 (8.9 mg)
BRB2 (3.0 mg)
Scheme 7 Separation of the butanol extract of the roots of Bauhinia sirindhorniae
85
Fr. RB-D (790.2 mg)
Silica gel
CHCl3: MeOH: H2O
(8:2:0.5)
Fr. RB-D1
(166.9 mg)
Fr. RB-D2
(78.5 mg)
Sephadex LH 20
CHCl3: MeOH
(1:1)
Fr. RB-D1-1
(34.5 mg)
Fr. RB-D1-2 Fr. RB-D2-1
(21.5 mg)
(106.3 mg)
Cosmosil
MeOH: H2O
(1:4)
BRB5 (30.0 mg)
Sephadex LH 20
CHCl3: MeOH
(1:1)
Fr. RB-D2-2
(18.3 mg)
Fr. RB-D4
(196.3 mg)
Fr. RB-D3
(168.2 mg)
Fr. RB-D2-3 Fr. RB-D2-4
(19.8 mg)
(8.9 mg)
Recrystallization
BRB6 (1.0 mg)
Sephadex LH 20
CHCl3: MeOH
(1:1)
Fr. RB-D3-1
(45.7 mg)
Fr. RB-D3-2
(63.7 mg)
ODS HPLC
MeOH: H2O
(2.5:8.5)
BRB7 (3.2 mg)
Scheme 8 Separation of fraction RB-D from the butanol extract of the roots of Bauhinia sirindhorniae
86
EtOAc Extract (110.7 g) from leaves of Croton hutchinsonianus
Vacuum Liquid column chromatography
Silica gel, Hexane: EtOAc
Fr. LE-A
(4.3 g)
Fr. LE-B
(20.2 g)
Silica gel
Hexane: EtOAc
(98:2)
Fr. LE-A1
(1.5 g)
Fr. LE-A2
(33.5 mg)
Fr. LE-A3
(2.7 g)
Sephadex LH 20
CH2Cl2: acetone
(1:1)
Fr. LE-B1
(9.2 g)
Fr. LE-B2-1
(1.7 g)
Fr. LE-C1
(39.2 g)
Fr. LE-B2
(10.1 g)
Fr. LE-B2-2
(98.9 mg)
Fr. LE-D
(25.8 g)
Sephadex LH 20
CH2Cl2: acetone
(1:1)
Cosmosil
MeOH: H2O (9:1)
PTLC
CBE1 (19.8 mg)
Fr. LE-C
(48.6 g)
Fr. LE-B2-3
(7.3 g)
Fr. LE-C2
(5.8 g)
PTLC
CBE4 (98.5 mg)
CBE2 (48.8 mg)
Scheme 9 Separation of the ethyl acetate extract of the leaves of Croton hutchinsonianus
87
EtOAc Extract (20.7 g) from branches of Croton hutchinsonianus
Vacuum Liquid column chromatography
Silica gel, Hexane: EtOAc
Fr. BE-A
(1.2 g)
Fr. BE-B
(3.5 g)
Sephadex LH 20
CH2Cl2: acetone
(1:1)
Silica gel
Hexane: EtOAc
(98:2)
Fr. BE-A1
(19.2 mg)
Fr. BE-A2
(21.4 mg)
Fr. BE-A3
(48.9 mg)
Fr. BE-B1
(1.2 g)
Fr. BE-B2-1
(47 mg)
Fr. BE-B2
(1.1 g)
Fr. BE-B2-2
(25.6 mg)
Fr. BE-D
(6.9 g)
Sephadex LH 20
CH2Cl2: acetone
(1:1)
Fr. BE-C2
(1.3 g)
Fr. BE-C1
(3.4 g)
Cosmosil
MeOH: H2O (9:1)
PTLC
CBE1 (2.1 mg)
Fr. BE-C
(5.7 g)
Fr. BE-B2-3
(36.9 g)
Recrystallization
Fr. BE-C2-1
(27.2 mg)
Silica gel
Hexane: EtOAc
(3:2)
Fr. BE-C2-2
(45.3 mg)
Fr. BE-C2-3
(11.5 mg)
PTLC
CBE3
(11.6 mg)
CBE2 (5.7 mg)
PTLC
CBE5
(18.9 mg)
CBE4
(35.6 mg)
Scheme 10 Separation of the ethyl acetate extract of the branches of Croton hutchinsonianus
PTLC
CBE6
(4.9 mg)
88
HO
HO
BSC1 [77]
BSC2 [214]
MeO
OH
OH
O-Rhamnose
HO
HO
OMe
OH
OH
O
BSB1 [14]
BSB2 [215]
OH
OH
OMe
HO
O
OMe
OH
OMe
Glucose-O
BSB3 [216]
OH
BSB4 [217]
NC
COOH
Glucose-O
OH
OH
BSB5 [218]
HO
OH
BSB6 [54]
Figure 3 Structures of compounds isolated from the stems of Bauhinia sirindhorniae
89
OH
HO
O
O
Glucose-O
BRC1 [219]
BRC2 [37]
OH
R
OH
OH
HO
HO
O
OH
O
O
OH
BRB1 [17]; R = H
O
BRB2 [220]
BRB3 [16]; R = OH
MeO
OH
O-Rhamnose
OH
OH
HO
OMe
O
OH
OH
HO
MeO
O
OMe
OH
BRB4 [221]
BRB5 [222]
NC
OH
Glucose-O
O
O
BRB6 [223]
Glucose-O
OH
BRB7 [224]
Figure 4 Structures of compounds isolated from the roots of Bauhinia sirindhorniae
90
O
COOH
CBE1 [225]
CBE2 [163]
O
R1
O
O
H
OH
R2
HO
CBE3 [226]
CBE4 [227]; R1 = OMe, R2 = OMe
CBE5 [228]; R1 = OMe, R2 = H
CBE6 [229]; R1, R2 = H
Figure 5 Structures of compounds isolated from the leaves and branches of
Croton hutchinsonianus
91
4. Physical and Spectral data of Isolated Compounds
4.1 Compound BSC1 (Lupeol)
Compound BSC1 was obtained as a colorless needle and found to be soluble
in chloroform (5.7 mg, 1.6×10-3% base on dried weight of stems).
: m/z (% relative intensity); Figure 11
EIMS
426 (M+, 48), 408 (100), 218 (78), 207 (30), 203 (50), 189
(75), 135 (66), 121 (71), 107 (69)
: νmax cm-1, KBr disc; Figure 10
IR
3447, 2927, 1650, 1457, 1386
1
H NMR
13
C NMR
: δ ppm, 500 MHz, in chloroform-d; Figure 12, Table 8
: δ ppm, 125 MHz, in chloroform-d; Figure 13, Table 8
4.2 Compound BSC2 (Glutinol)
Compound BSC2 was obtained as a colorless needle and found to be soluble
in chloroform (16.3 mg, 4.7×10-3% base on dried weight of stems).
: m/z (% relative intensity); Figure 15
EIMS
426 (M+, 20), 408 (100), 274 (98), 259 (76), 173 (63),
161 (58)
: νmax cm-1, KBr disc; Figure 14
IR
3461, 2933, 1455, 1385, 1037, 971, 800
1
H NMR
13
C NMR
: δ ppm, 500 MHz, in chloroform-d; Figure 16, Table 9
: δ ppm, 125 MHz, in chloroform-d; Figure 17, Table 9
4.3 Compound BSB1 (Isoliquiritigenin)
Compound BSB1 was obtained as a yellow crystal and found to be soluble in
DMSO (4.5 mg, 1.3×10-3% base on dried weight of stems).
FAB+MS
: [M+H] + at m/z 257 (positive ion mode); Figure 20
UV
: λ max nm (log ε) in methanol; Figure 18
365 (4.30)
IR
: νmax cm-1, KBr disc; Figure 19
3301, 1635, 1604, 1564, 1513, 1367, 1294, 1219, 1175, 1128,
1033, 978, 891, 826, 802, 621, 558, 524
Melting point
: 182-183 °C
1
: δ ppm, 400 MHz, in DMSO-d6; Figure 21, Table 10
H NMR
13
C NMR
: δ ppm, 100 MHz, in DMSO-d6; Figure 22, Table 10
92
4.4 Compound BSB2 ((+)-Isolariciresinol-3α-O-α-L-rhamnoside)
Compound BSB2 was obtained as an amorphous powder and found to be
soluble in methanol (2.0 mg, 5.7×10-4% base on dried weight of stems).
FAB+MS
: [M+H] + at m/z 507 (positive ion mode); Figure 28
UV
: λ max nm (log ε) in methanol; Figure 26
221 (4.61), 283 (4.16)
: νmax cm-1, KBr disc; Figure 27
IR
3401, 2932, 1602, 1515, 1455, 1380, 1253, 1129, 1049, 879
[α] D23
: +20.8° (methanol, c 0.25)
1
: δ ppm, 500 MHz, in methanol-d4; Figure 29, Table 11
H NMR
13
C NMR
: δ ppm, 125 MHz, in methanol-d4; Figure 30, Table 11
4.5 Compound BSB3 (3,4,5-Trimethoxyphenolic-1-O-β-D-glucoside)
Compound BSB3 was obtained as a white needle and found to be soluble in
methanol (3.8 mg, 1.1×10-3% base on dried weight of stems).
FAB+MS
: [M+H] + at m/z 347 (positive ion mode); Figure 33
UV
: λ max nm (log ε) in methanol; Figure 31
222 (4.39), 268 (4.01), 288 (3.92)
: νmax cm-1, KBr disc; Figure 32
IR
: 3404, 1697, 1614, 1515, 1288, 1072, 763
Melting Point
: 199-202 °C
1
: δ ppm, 500 MHz, in methanol-d4; Figure 34, Table 12
H NMR
13
C NMR
: δ ppm, 125 MHz, in methanol-d4; Figure 35, Table 12
4.6 Compound BSB4 ((-)-Epicatechin)
Compound BSB4 was obtained as a colorless needle and found to be soluble
in methanol (8.5 mg, 2.4×10-3% base on dried weight of stems).
FAB-MS
: [M-H] - at m/z 289 (negative ion mode); Figure 40
UV
: λ max nm (log ε) in methanol; Figure 38
222 (4.96), 280 (4.31)
IR
: νmax cm-1, KBr disc; Figure 39
3459, 2932, 1625, 1552, 1442, 1261, 1145, 808, 795
[α] D23
: -55° (methanol, c 0.25)
93
: [θ]219 –11100.6, [θ]240 +4017.9; [θ]280 –1614.6 (c 3.2 × 10-4,
CD
methanol) 23 oC
Melting Point
: 235-237 °C
1
: δ ppm, 500 MHz, in methanol-d4; Figure 41, Table 13
H NMR
13
C NMR
: δ ppm, 125 MHz, in methanol-d4; Figure 42, Table 13
4.7 Compound BSB5 (Protocatechuic acid)
Compound BSB5 was obtained as a colorless crystal and found to be soluble
in methanol (8.0 mg, 2.3×10-3% base on dried weight of stems).
FAB+MS
: [M+H] + at m/z 155 (positive ion mode); Figure 48
UV
: λ max nm (log ε) in methanol; Figure 46
222 (4.68), 258 (4.47), 294 (4.23)
: νmax cm-1, KBr disc; Figure 47
IR
: 3264, 1673, 1601, 1297, 943, 766, 559
Melting Point
: 194-196 °C
1
: δ ppm, 500 MHz, in methanol-d4; Figure 49, Table 14
H NMR
13
C NMR
: δ ppm, 125 MHz, in methanol-d4; Figure 50, Table 14
4.8 Compound BSB6 (Lithospermoside)
Compound BSB6 was obtained as a fine white needle and found to be soluble
in water (5.5 mg, 1.6×10-3% base on dried weight of stems).
FAB+MS
: [M+H] + at m/z 330 (positive ion mode); Figure 53
UV
: λ max nm (log ε) in water; Figure 51
259 (3.84)
: νmax cm-1, KBr disc; Figure 52
IR
3434, 2914, 2224, 1601, 1379, 1256, 1080, 1045, 997, 948,
849, 654
CD
: [θ]263 -44030, [θ]227 +35086; (c 3.1 × 10-4, water) 23oC
1
: δ ppm, 500 MHz, in water-d2; Figure 54, Table 15
H NMR
13
C NMR
: δ ppm, 125 MHz, in water-d2; Figure 55, Table 15
4.9 Compound BRC1 (5,7-Dihydroxychromone)
Compound BRC1 was obtained as a colorless needle and found to be soluble
in methanol (7.2 mg, 2.4×10-3% base on dried weight of roots).
FAB+MS
: [M+H] + at m/z 179 (positive ion mode); Figure 60
94
: λ max nm (log ε) in methanol; Figure 58
UV
224 (4.83), 250 (4.93), 256 (4.95), 295 (4.53)
: νmax cm-1, KBr disc; Figure 59
IR
: 3003, 2731, 2628, 1646, 1617, 1500, 1373, 1187, 1032, 845
1
H NMR
: δ ppm, 500 MHz, in methanol-d4; Figure 61, Table 16
: δ ppm, 500 MHz, in acetone-d6; Figure 62
13
C NMR
: δ ppm, 125 MHz, in methanol-d4; Figure 63, Table 16
4.10 Compound BRC2 (Sitosteryl-3-O-β-D-glucoside)
Compound BRC2 was obtained as a white powder and found to be soluble in
chloroform in methanol (10.3 mg, 3.4×10-3% base on dried weight of roots).
FAB+MS
: [M+Na] + at m/z 577 (positive ion mode); Figure 67
IR
: νmax cm-1, KBr disc; Figure 66
: 3402, 2934, 1463, 1367, 1168, 1073, 1025, 802
1
H NMR
: δ ppm, 500 MHz, in methanol-d4 + chloroform-d; Figure 68,
Table 17
13
C NMR
: δ ppm, 125 MHz, in methanol-d4 + chloroform-d; Figure 69,
Table 17
4.11 Compound BRB1 ((2S)-Naringenin)
Compound BRB1 was obtained as a pale yellow needle and found to be
soluble in methanol (3.7 mg, 1.2×10-3% base on dried weight of roots).
FAB+MS
: [M+H] + at m/z 273 (positive ion mode); Figure 72
UV
: λ max nm (log ε) in methanol; Figure 70
226 (4.75), 288 (4.57), 332 (3.91)
: νmax cm-1, KBr disc; Figure 71
IR
: 3268, 1632, 1604, 1463, 1253, 1158, 1084, 832, 728
[α] D23
: -13° (MeOH, c 0.23)
Melting Point
: 249-251 °C
1
: δ ppm, 500 MHz, in methanol-d4; Figure 73, Table 18
H NMR
13
C NMR
: δ ppm, 125 MHz, in methanol-d4; Figure 74, Table 18
4.12 Compound BRB2 (Luteolin)
Compound BRB2 was obtained as a yellow needle and found to be soluble in
DMSO (3.0 mg, 1.0×10-3% base on dried weight of roots).
95
FAB+MS
: [M+H] + at m/z 287 (positive ion mode); Figure 79
UV
: λ max nm (log ε) in methanol; Figure 77
221 (4.84), 255 (4.74), 267 (4.71), 350 (4.81)
: νmax cm-1, KBr disc; Figure 78
IR
: 3395, 1657, 1608, 1510, 1360, 1259, 1167, 1031, 838, 641
Melting Point
: 325-328 °C
1
: δ ppm, 500 MHz, in; Figure 80, Table 19
H NMR
13
C NMR
: δ ppm, 125 MHz, in; Figure 81, Table 19
4.13 Compound BRB3 ((2S)-Eriodictyol)
Compound BRB3 was obtained as a pale yellow needle and found to be
soluble in methanol (7.3 mg, 2.4×10-3% base on dried weight of roots).
FAB+MS
: [M+H] + at m/z 289 (positive ion mode); Figure 86
UV
: λ max nm (log ε) in methanol; Figure 84
224 (4.98), 288 (4.91), 328 (4.21)
: νmax cm-1, KBr disc; Figure 85
IR
: 3366, 1632, 1605, 1452, 1311, 1086, 825, 735
[α] D23
: -10° (methanol, c 0.39)
Melting Point
: 250-153 °C
1
: δ ppm, 500 MHz, in methanol-d4; Figure 87, Table 20
H NMR
13
C NMR
: δ ppm, 125 MHz, in methanol-d4; Figure 88, Table 20
4.14 Compound BRB4 ((+)-Taxifolin)
Compound BRB4 was obtained as a pale yellow needle and found to be
soluble in methanol (8.9 mg, 2.5×10-3% base on dried weight of roots).
FAB+MS
: [M+H] + at m/z 305 (positive ion mode); Figure 93
UV
: λ max nm (log ε) in methanol; Figure 91
222 (4.99), 290 (4.91), 325 (4.39)
IR
: νmax cm-1, KBr disc; Figure 92
: 3412, 1639, 1615, 1476, 1265, 1083, 808, 780
[α] D23
: +17° (methanol, c 0.32)
CD
: [θ]329 +10200.5, [θ]299 –4100.2; (c 3.1 × 10-4, methanol) 23 oC
Melting Point
: 238-241 °C
1
: δ ppm, 500 MHz, in methanol-d4; Figure 93, Table 21
H NMR
96
13
C NMR
: δ ppm, 125 MHz, in methanol-d4; Figure 94, Table 21
4.15 Compound BRB5 ((+)-Lyoniresinol-3α-O-α-L-rhamnoside)
Compound BRB5 was obtained as an amorphous solid and found to be soluble
in methanol (30.0 mg, 1.0×10-2% base on dried weight of roots).
FAB+MS
: [M+K] + at m/z 605 (positive ion mode); Figure 100
UV
: λ max nm (log ε) in methanol; Figure 98
221 (4.85), 278 (3.96)
: νmax cm-1, KBr disc; Figure 99
IR
3402, 2937, 1614, 1517, 1461, 1056, 982, 83, 809, 637
[α] D23
: +3.3° (methanol, c 0.50)
1
: δ ppm, 500 MHz, in methanol-d4; Figure 101, Table 22
H NMR
13
C NMR
: δ ppm, 125 MHz, in methanol-d4; Figure 102, Table 22
4.16 Compound BRB6 (5-Hydroxychromone-7-β-D-glucoside)
Compound BRB6 was obtained as a white needle and found to be soluble in
methanol (1.0 mg, 3.0×10-4% base on dried weight of roots).
FAB+MS
: [M+H] + at m/z 341 (positive ion mode); Figure 107
UV
: λ max nm (log ε) in methanol; Figure 106
221 (4.06), 252 (4.01), 256 (4.03), 288 (3.42)
1
H NMR
13
C NMR
: δ ppm, 500 MHz, in methanol-d4; Figure 108, Table 23
: δ ppm, 125 MHz, in methanol-d4; Figure 109, Table 23
4.17 Compound BRB7 (Menisdaurin)
Compound BRB7 was obtained as a white powder and found to be soluble in
methanol (3.2 mg, 1.1×10-3% base on dried weight of stems).
FAB+MS
: [M+H] + at m/z 314, [M+Na] + at m/z 336, [M+K] + at m/z 352
(positive ion mode); Figure 112
UV
: λ max nm (log ε) in methanol; Figure 110
258 (4.82)
IR
: νmax cm-1, KBr disc; Figure 111
3404, 2912, 2218, 1520, 1456, 1266, 1044, 843
[α] D23
: -195° (methanol, c 1.0)
1
: δ ppm, 500 MHz, in methanol-d4; Figure 113, Table 24
H NMR
13
C NMR
: δ ppm, 125 MHz, in methanol-d4; Figure 114, Table 24
97
4.18 Compound CBE1 (Farnesyl acetone)
Compounds CBE1 (19.8 mg, 7.9×10-4% base on dried weight of leaves and
2.1 mg, 1.8×10-4% base on dried weight of branches) was obtained as a colorless oil
and found to be soluble in chloroform.
: m/z (% relative intensity); Figure 119
EIMS
262 (M+, 32), 245 (100), 243 (17), 201 (14), 189 (14), 175
(15), 163 (22), 161 (16), 137 (15), 121 (28), 109 (14), 95 (18)
: νmax cm-1, neat; Figure 118
IR
3019, 2974, 2400, 1712, 1221, 762, 730, 457
1
H NMR
13
C NMR
: δ ppm, 400 MHz, in chloroform-d; Figure 120, Table 25
: δ ppm, 100 MHz, in chloroform-d; Figure 121, Table 25
4.19 Compound CBE2 (Poilaneic acid)
Compounds CBE2 (48.8 mg, 2.0×10-3% base on dried weight of leaves and
25.6 mg, 2.0×10-3% base on dried weight of branches) was obtained as a colorless
needle and found to be soluble in chloroform.
: m/z (% relative intensity); Figure 127
EIMS
302 (M+, 15), 287 (19), 284 (14), 259 (40), 257 (37), 241 (24),
213 (39), 185 (30), 171 (30), 157 (32), 143 (32), 133 (26), 129
(34), 121 (26), 119 (34), 107 (30), 105 (69), 93 (37), 91 (100),
87 (63), 79 (50), 77 (55), 55 (26)
: λ max nm (log ε) in methanol; Figure 125
UV
230 (4.62)
: νmax cm-1, neat; Figure 126
IR
3445, 2917, 2849, 1699, 1458, 1262, 1033
[α] D23
: -140° (chloroform, c 0.25)
1
: δ ppm, 500 MHz, in chloroform-d; Figure 128, Table 26
H NMR
13
C NMR
: δ ppm, 125 MHz, in chloroform-d; Figure 129, Table 26
4.20 Compound CBE3 (4-Hydroxybenzaldehyde)
Compound CBE3 was obtained as a colorless needle and found to be soluble
in chloroform (11.6 mg, 9.7×10-4% base on dried weight of branches).
EIMS
: m/z (% relative intensity); Figure 136
122 (10), 121 (100), 105 (16), 93 (15), 77 (15), 74 (11), 66
(5), 65(24), 63 (18), 62 (24), 61 (12)
98
: λ max nm (log ε) in methanol; Figure 134
UV
222 (4.12), 284 (4.24)
: νmax cm-1, KBr disc; Figure 135
IR
3164, 1666, 1597, 1454, 1286, 1217, 1160, 835, 705
Melting point
: 113-115 °C
1
: δ ppm, 400 MHz, in chloroform-d; Figure 138, Table 27
H NMR
13
C NMR
: δ ppm, 100 MHz, in chloroform-d; Figure 139, Table 27
4.21 Compound CBE4 (3-(4-Hydroxy-3,5-dimethoxyphenyl)-propyl benzoate)
Compounds CBE4 (98.5 mg, 3.9×10-3% base on dried weight of leaves and
35.6 mg, 3.0×10-3% base on dried weight of branches) was obtained as a pale yellow
oil and found to be soluble in chloroform.
HREIMS
: [M+H] + at m/z 317.1395 calcd for C18H20O5, 317.1389
EIMS
: m/z (% relative intensity); Figure 144
316 (M+, 100), 194 (84), 163 (75), 105 (30), 77 (76)
: λ max nm (log ε) in methanol; Figure 142
UV
228 (3.85), 272 (2.89)
: νmax cm-1, neat; Figure 143
IR
3446, 2921, 1708, 1520, 1300, 1213, 1112, 712
1
H NMR
13
C NMR
: δ ppm, 400 MHz, in chloroform-d; Figure 145, Table 28
: δ ppm, 100 MHz, in chloroform-d; Figure 146, Table 28
4.22 Compound CBE5 (Dihydroconiferyl benzoate)
Compound CBE5 was obtained as a pale yellow oil and found to be soluble in
chloroform (18.9 mg, 1.6×10-3% base on dried weight of branches).
HRFABMS
: [M+H] + at m/z 287.1289 calcd for C17H18O4, 287.1284
EIMS
: m/z (% relative intensity); Figure 153
286 (M+, 100), 164 (100), 133 (34), 105 (23), 77 (36)
UV
: λ max nm (log ε) in methanol; Figure 151
229 (4.35), 280 (3.66)
IR
: νmax cm-1, neat; Figure 152
3428, 2957, 1718, 1604, 1516, 1273, 1119, 712
1
H NMR
13
C NMR
: δ ppm, 400 MHz, in chloroform-d; Figure 154, Table 29
: δ ppm, 100 MHz, in chloroform-d; Figure 155, Table 29
99
4.23 Compound CBE6 (3-(4-Hydroxyphenyl)-propyl benzoate)
Compound CBE6 was obtained as a pale yellow oil and found to be soluble in
chloroform (4.9 mg, 4.1×10-4% base on dried weight of branches).
HRFABMS
: [M+H] + at m/z 257.1179 calcd for C16H16O3, 257.1178
EIMS
: m/z (% relative intensity); Figure 161
258 (M+, 3), 134 (38), 133 (100), 105 (50), 103 (17), 77 (36)
: λ max nm (log ε) in methanol; Figure 159
UV
228 (4.08), 279 (3.26)
: νmax cm-1, neat; Figure 160
IR
3377, 1698, 1633, 1516, 1277, 1118, 712
1
: δ ppm, 400 MHz, in chloroform-d; Figure 162, Table 30
H NMR
13
: δ ppm, 100 MHz, in chloroform-d; Figure 163, Table 30
C NMR
5. Evaluation of Biological Activities
5.1 Antimicrobial Activity
5.1.1 Agar Diffusion Assay
Antimicrobial activity of the crude extracts were screened by agar diffusion
method (Jorgensen et al, 1999; Ingroff et al., 1999).
The bacterial strains used were as follows:
-
Staphylococcus aureus ATCC 29213
-
Bacillus subtilis ATCC 6633
-
Pseudomonas aeruginosa ATCC 27853
-
Escherichia coli ATCC 25922
The fungal strains used were as follows:
-
Candida albicans ATCC 10231
-
Trichophyton mentagrophytes (clinical isolated)
5.1.1.1 Preparation of Sample
The amounts of crude extracts were 10 mg per disk.
5.1.1.2 Preparation of the Inoculum
Each bacterial strain was cultured overnight on trypticase soy agar (TSA) plate
at 37 °C. The isolated colonies were inoculated into a 5 ml trypticase soy broth (TSB)
and incubated at 37 °C for 2-3 hours. The turbidity of these inocula was adjusted to
match that of a 0.5 McFarland standard (approximately 108 CFU/ml for bacteria).
100
Candida albicans ATCC 10231 was grown on Sabouraud dextrose agar
(SDA) slant at 30 °C for 24 hours. The inoculum was prepared by suspending the
culture in sterile normal saline solution and turbidity of the inoculum was adjusted to
match a 0.5 turbidity standard of McFarland.
Spores of Trichophyton mentagrophytes grown on SDA slant at 30 °C for five
days were washed from the slant culture with sterile 0.05% Tween 80. The turbidity
of the spore suspension was adjusted to match 0.5 turbidity standard of McFarland
(this produced a fungal suspension containing 1×106 to 5×106 organisms per ml).
5.1.1.3 Preparation of Test Plates
5.1.1.3.1 Preparation for Testing Bacteria
Mueller Hinton agar (MHA) was melted and allowed to cool at 45-50 °C in a
water bath. Then 25 ml of the melted agar medium was dispensed into sterile glass
petri dishes, with internal diameters of 9 cm, to yield a uniform depth of 4 mm. The
agar was allowed to harden on a flat level surface. The plates were dried for 1 hour at
37 °C.
5.1.1.3.2 Preparation for Testing Fungi
Sabouraud dextrose agar (SDA) was used and prepared as described above.
5.1.1.4 Inoculation of Agar Plates
A sterile cotton swab was dipped in each inoculum and the excess was
removed by rotating the swab several times against the inside wall of the tube above
the fluid level. The entire surfaces of the MHA plate and the SDA plate for testing
bacteria and fungi, respectively, were inoculated by streaking with the swab for three
times and each time the plate was rotated 60 degree.
5.1.1.5 Assay Procedure
Within 15 minutes after the plates were inoculated, the sample disks were
placed individually then gently pressed down onto the agar surface. This was done in
duplicate. After maintaining at room temperature for 1 hour, the bacterial and fungal
plates were incubated at 37 °C overnight and 30 °C for 48-72 hours, respectively. The
sample disks showing inhibition zone were examined further for their minimal
inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC).
101
5.1.2 Determination of MIC and MBC
Determination of the MIC and MBC of pure compounds against
Staphylococcus aureus ATCC 29213 and Bacillus subtilis ATCC 6633 by broth
microdilution test (Jorgensen et al., 1999).
5.1.2.1 Preparation of Test Samples
The samples were dissolved in DMSO and diluted with Mueller Hinton broth
(MHB) in a two-fold dilution to give the concentrations ranging from 200 µg/ml to
0.39 µg/ml.
5.1.2.2 Preparation of the Inoculum
The inoculum was prepared as described in 5.1.1.2. The inoculum was further
diluted to 1:100 in MHB.
5.1.2.3 Assay Procedure
A 50 µl volume of each concentration of the sample was dispensed to the
corresponding well of the sterile multiwell microdilution plate (96-Flat-shaped wells).
Another 50 µl volume of diluted inoculum was added into each well.
After
incubating the tray at 37 °C for 24 hours, the lowest concentration of the sample that
showed growth inhibition was considered as the MIC. This determination was done
in duplicate.
All inhibitory concentrations were re-checked by addition of each
solution showing activity into agar plate, and incubated at 37 °C for 24 hours. The
lowest concentration of the test compounds which kill these microorganisms were
defined as MBC. Penicillin G was used as a positive control.
5.2 Determination of Free Radical Scavenging Activity
5.2.1 TLC Screening Assay (Pezzuto and Kinghorn, 1998)
Free radical scavenging activity of the crude extracts were screened by TLC
screening method. The samples were spotted and developed on a TLC plate with
suitable developing solvent. After drying, the TLC plate was sprayed with 80 µg/ml
solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH) in methanol.
examined 30 minutes after spraying.
The plate was
Active compounds appear as yellow spots
against purple background.
5.2.2 Free Radical Scavenging Activity Assay (Takao et al., 1994; BrandWilliams, Cuvelier, and Berset, 1995)
102
5.2.2.1 Preparation of the Test Sample
Compounds BSB2 [216], BSB6 [54], BRB5 [223] and BRB7 [225] from B.
sirindhorniae were first tested at 40 µg/ml. Compounds exhibiting more than 50%
inhibition were further analyzed for their IC50 values. Each test sample was prepared
as an ethanolic solution with initial concentration of 80 µg/ml. For analysis serial
dilution was performed to give seven concentrations (40 µg/ml, 20 µg/ml, 10 µg/ml, 5
µg/ml, 2.5 µg/ml, 1.25 µg/ml and 0.625 µg/ml). Assays were carried out in duplicate.
The test sample (100 µl) was added to the reaction mixture (100 µl) to furnish the
total volume of 200 µl. The final concentration was calculated by the formula below.
N1V1 = N2V2
N1 = Initial concentration (µM)
V1 = Initial volume (µl)
N2 = Final concentration (µM)
V2 = Final volume (µl)
For example, of test sample (80 µg/ml) was added to the reaction mixture to
furnish the total volume of 200 µl.
Thus, final concentration of test sample = 80 µg/ml×100 µl/200 µl
= 40 µg/ml
The initial and final concentrations (µg/ml) of test sample
Initial concentration (µg/ml)
80
40
20
10
5
2.50 1.250 0.625
Final concentration (µg/ml)
40
20
10
5
2.5
1.25 0.625 0.312
5.2.2.2 Preparation of the DPPH Solution (200 µM)
1,1-Diphenyl-2-picrylhydrazyl (DPPH) 7.88 mg was dissolved in ethanol
100 ml and the solution (200 µM) was subsequently stirred for 30 minutes.
103
5.2.2.3 Measurement of Activity
N
N
N
+ AH
.
O2 N
NO2
+ A.
NH
O2 N
NO2
.
DPPH
(Purple)
NO2
NO2
DPPH-H
(Pale yellow)
DPPH = 1,1-diphenyl-2-picrylhydrazyl
AH = antioxidant
The test sample (100 µl) was dissolved in ethanol and mixed with 200 µM
DPPH ethanolic solution (100 µl) in a 96-well microtiter plate. The reaction mixture
was shaken well and kept in the dark for 20 minutes. The absorbance at 515 nm was
measured by microtiter plate reader (Biorad, model 550). The DPPH solution was
used as a negative control. Vitamin E derivative Trolox was used as a standard
control and quercetin was used as a positive control. The decrease in absorbance per
µM of each sample was compared with that of Trolox.
5.3 Cytotoxic Activity
Compounds CBE4 [227], CBE5 [228] and CBE6 [229] were determined for
cytotoxicity by employing the colorimetric method against NCI-H187 (human small
cell lung cancer cell line) using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) method (Skehan et al., 1990). The IC50 values
of the tested compounds were measured in µg/ml. Ellipticine was used as a positive
control, exhibiting the activity with the IC50 of 0.35 µg/ml.
5.4 Antifungal Activity
Compounds CBE4 [227], CBE5 [228] and CBE6 [229] were evaluated for
antifungal activity against Candida albicans, employing the colorimetric method
(Hawser et al., 1998). The IC50 values of the tested compounds were measured in
µg/ml. Amphotericin B was used as a positive control, exhibiting the activity with the
IC50 of 0.02 µg/ml.
CHAPTER IV
RESULTS AND DISCUSSION
The pulverized stems and roots of Bauhinia sirindhorniae K & S.S. Larsen
were successively extracted with hexane, chloroform, and 95% ethanol. The 95%
ethanol extracts were investigated by several chromatographic techniques to give
seven-teen compounds classified as two cyanoglucosides (BSB6 and BRB7), one
flavan (BSB4), two flavanones (BRB1 and BRB3), one flavanonol (BRB4), one
flavone (BRB2), one chalcone (BSB1), one chromone (BRC1), one chromone
glucoside (BRB6), two lignan glycosides (BSB2 and BRB5), two triterpenoids (BSC1
and BSC2), one steroid glucoside (BRC2) and other phenolic compounds (BSB3 and
BSB5). The antimicrobial and free radical scavenging activities of some compounds
were evaluated.
The dried leaves and branches of Croton hutchinsonianus Hosseus. were
dried, grounded and then sequentially percolated with hexane, ethyl acetate and 95%
ethanol, respectively. After successive extraction, the solvents were removed under
reduced pressure. The last trace of solvents were eliminated under high vacuum to
afford gums which were submitted for cytotoxic assays. Cytotoxic of various extracts
are demonstrated result in Table 33.
The hexane and ethyl acetate extract of the leaves and branches showed
cytotoxic activity against NCI H-187 cell lines as shown in Table 33. The ethyl
acetate extract of the leaves was firstly separated by repeated column chromatography
to give one C18 terpenoid (CBE1), one diterpene (CBE2) and one phenylpropyl
benzoate (CBE4). The chemical investigation of the ethyl acetate extract of the
branches has led to the isolation of the same three compounds (CBE1, CBE2 and
CBE4), together with one benzaldehyde (CBE3) and two phenylpropyl benzoates
(CBE5 and CBE6). No pure compound was isolated from hexane and 95% ethanol
extract of the leaves and branches.
The structures of all isolates were determined based on their UV, IR, MS and
NMR data, and then discussed by the comparison with the literature values.
105
1. Structure Determination of Isolated Compounds
1.1 Structure Determination of Compound BSC1
29
21
19
20
18
12
22
13
11
25
17
26
28
1
14
16
9
10
15
8
27
4
7
5
6
30
2
3
HO
24
23
[77]
Compound BSC1 was obtained as a colorless needle. It showed a molecular
[M+] ion peak at m/z 426 in EIMS (Figure 11), suggesting a molecular formula of
C30H50O. The fragmentation ions in the mass spectrum of compound BSC1 at m/z
426 [M+] were useful in obtaining the structure of compound BSC1 and were in
agreement with those reported in the literature (Hui and Fung, 1969; Hui and Lee,
The ions at m/z 408 could reasonably come from [M+-H2O].
1971).
fragmentation pathways are as shown in Scheme 11.
Other
The IR spectrum of this
compound showed O-H stretching broad band at 3447 cm-1 which indicated the
presence of hydroxy group (Figure 10).
The 1H NMR spectrum in CDCl3 (Figure 12 and Table 8) displayed signals for
seven methyl groups at δ 0.76, 0.77, 0.85, 0.96, 0.98, 1.04 and 1.66. Signals for
several methine and methylene protons appeared at δ 0.90-1.80. In addition, a proton
signal at δ 3.19 (dd, J = 11.2, 4.6 Hz, H-3), a multiplet proton signal at δ 2.35 (H-19)
and two broad singlet proton signals at δ 4.54 and δ 4.66 (H-29) were also observed.
The 13C NMR spectrum in CDCl3 (Figure 13 and Table 8) showed 30 carbon signals,
corresponding to seven methyls, eleven methylenes, six methines and six quaternary
carbons.
These 1H and 13C NMR data were in good agreement with those reported for
lupeol [77] (Reynolds et al., 1986) as shown in Table 8.
106
+.
+.
E
E
C
D
B
A
H
D
HO
CH3
m/z 426 (48%)
m/z 218 (78%)
-H2O
+
m/z 408 (100%)
CH2
A
B
HO
m/z 207 (30%)
-H2O
m/z 189 (75%)
Scheme 11 EIMS Spectra fragmentations of compound BSC1
107
Table 8 NMR Spectral data of compound BSC1 and lupeol (in CDCl3)
Compound BSC1
Position
1
H (mult., J in Hz)
Lupeol
13
C
1
H (mult., J in Hz)
13
C
1a
0.90-1.80
38.7
1.68
38.6
1b
0.90-1.80
-
0.91
-
2a
0.90-1.80
27.4
1.61
27.3
2b
0.90-1.80
-
1.54
-
3
3.19 (dd, 11.2, 4.6)
79.0
3.18 (dd)
78.9
4
-
38.9
-
38.8
5
0.90-1.80
55.3
0.69
55.2
6a
0.90-1.80
18.3
1.54
18.2
6b
0.90-1.80
-
1.39
-
7
0.90-1.80
34.3
1.41
34.2
8
-
40.8
-
40.7
9
0.90-1.80
50.4
1.28
50.3
10
-
37.2
-
37.1
11a
0.90-1.80
20.9
1.42
20.9
11b
0.90-1.80
-
1.25
-
12a
0.90-1.80
25.2
1.68
25.0
12b
0.90-1.80
-
1.07
13
0.90-1.80
38.1
1.67
38.0
14
-
42.8
-
42.7
15a
0.90-1.80
27.5
1.71
27.4
15b
0.90-1.80
-
1.01
-
16a
0.90-1.80
35.6
1.49
35.5
16b
0.90-1.80
-
1.38
-
17
-
43.0
-
42.9
18
0.90-1.80
48.3
1.37
48.2
19
2.35 (m)
48.0
2.39
47.9
20
-
151.0
-
150.8
21a
0.90-1.80
29.9
1.93
29.8
21b
0.90-1.80
-
1.33
-
22a
0.90-1.80
40.0
1.42
39.9
22b
0.90-1.80
-
1.20
-
23
0.98
28.0
0.98
27.9
24
0.76
15.4
0.77
15.3
25
0.85
16.1
0.84
16.1
26
1.04
16.0
1.04
15.9
27
0.96
14.6
0.97
14.5
28
0.77
18.0
0.79
17.9
29a
4.54 (br s)
109.3
4.56
109.3
29b
4.66 (br s)
-
4.69
-
30
1.66 (s)
19.3
1.69
19.2
108
1.2 Structure Determination of Compound BSC2
30
29
19 20
27
12 18
11
9
1
2
HO
24
4
17
14
8
10
3
13
25
5
7
21
22
28
16
15
26
6
23
[214]
Compound BSC2 was obtained as a colorless needle. The EIMS exhibited [M+]
at m/z 426 (Figure 15), corresponding to a molecular formula of C30H50O. The IR
spectrum of this compound showed O-H stretching band at 3461 cm-1 which indicated
the presence of hydroxy group (Figure 14).
The 1H NMR spectrum of compound BSC2 in CDCl3 (Figure 16 and Table 9)
displayed signals for eight methyls groups at δ 0.88, 0.94, 0.98, 1.02, 1.07, 1.11, 1.14
and 1.20. Signals for several methine and methylene protons appeared at δ 0.88-1.98.
In addition, two broad singlet proton signals at δ 3.44 and δ 5.61 were also observed.
Other 1H-NMR assignments were illustrated in Table 9.
The 13C NMR spectrum of compound BSC2 in CDCl3 (Figure 17 and Table 9)
showed 30 carbons signals, corresponding to eight methyls, ten methylenes, five
methine and seven quarternary carbons. These
13
C NMR data and 1H NMR data
which were in good agreement with those reported for glutinol [214] (Carvalho and
Seita, 1993; Gaind et al., 1976) as shown in Table 9.
This compound is a relatively rare triterpenol that is believed to be an
intermediate in the biogenetic pathway to friedelin. The triterpenic ketone glutinone
(also called alnusenone), isolated from Alnus glutinosa (Betulaceae), was the first
compound of this class isolated from a natural source (Zhong, Waterman and Jeffreys,
1984). Glutinol (D:B-friedoolean-5-en-3β-ol), obtained by reduction of glutinone and
which structure was later determined, was isolated for the first time from a natural
source from Euphorbia royleana (Mahato, Das and Sahu, 1981).
109
Table 9 NMR Spectral data of compound BSC2 and glutinol (in CDCl3)
Compound BSC2
Position
1
H (mult., J in Hz)
Glutinol
13
C
13
C
1
0.88-1.98
18.2
18.2
2
0.88-1.98
27.8
27.8
3
3.44 (br s)
76.3
76.4
4
0.88-1.98
40.8
40.8
5
0.88-1.98
141.6
141.6
6
5.61 (br s)
122.1
122.1
7
0.88-1.98
23.6
23.6
8
0.88-1.98
47.4
47.4
9
0.88-1.98
34.8
34.8
10
0.88-1.98
49.7
49.7
11
0.88-1.98
34.6
34.6
12
0.88-1.98
30.3
30.4
13
0.88-1.98
39.3
39.3
14
0.88-1.98
37.8
37.8
15
0.88-1.98
32.1
32.1
16
0.88-1.98
36.0
36.0
17
0.88-1.98
30.1
30.1
18
0.88-1.98
43.0
43.0
19
0.88-1.98
35.1
35.1
20
0.88-1.98
28.2
28.3
21
0.88-1.98
33.1
33.1
22
0.88-1.98
38.9
39.0
23
0.88-1.20
28.9
29.0
24
0.88-1.20
25.4
25.5
25
0.88-1.20
16.2
16.2
26
0.88-1.20
19.6
19.6
27
0.88-1.20
18.4
18.4
28
0.88-1.20
32.0
32.1
29
0.88-1.20
34.5
34.5
30
0.88-1.20
32.4
32.4
110
1.3 Structure Determination of Compound BSB1
3
HO 4'
2
1
5'
6'
β
α
1'
3'
2'
OH
OH
4
5
6
β'
O
[14]
Compound BSB1 was isolated as a yellow crystal with m.p. 182-183°C. The
structure of compound BSB1 was elucidated by spectroscopic methods. Its molecular
formula C15H12O4 was established by FAB+MS with the molecular ion [M+H]+ at m/z
257 (Figure 20), suggesting ten degrees of unsaturation.
The IR spectrum of
compound BSB1 exhibited characteristic absorption bands for hydroxyl (3514 cm-1)
and carbonyl (1634 cm-1) functionalities (Figure 19). The UV spectrum showed a
maximum absorption at 365 nm (Figure 18).
The
13
C NMR of compound BSB1 in DMSO-d6 (Figure 22 and Table 10)
exhibited 13 signals. The DEPT spectrum established the existence of nine methine
carbons, and six quaternary carbons as shown in Table 10.
The 1H NMR of compound BSB1 in DMSO-d6 (Figure 21 and Table 10)
showed three protons belonging to 1,2,4-trisubstituted benzene ring system (ABX
system ) at δ 8.15 (d, J = 8.8 Hz, H-6′), 6.39 (dd, J = 8.8, 2.4 Hz, H-5′), 6.26 (d, J =
2.4 Hz, H-3′). Two doublets at δ 7.73 (J = 16.0 Hz) and 7.76 (J = 16.0 Hz) were
observed, suggesting the presence of a double bond between C-α and C-β.
In
addition, four protons belonging to 1,4 disubstituted benzene ring system (AA′BB′
system) at δ 7.74 (d, J = 8.7 Hz, H-2 and H-6) and 6.83 (d, J = 8.7 Hz, H-3 and H-5)
were noted.
Correlations of these protons were observed in the 1H-1H COSY
spectrum (Figure 23). Connectivity of C-H bond and the connectivity of C-H through
two or three bond correlations were shown in the HMQC and HMBC spectrum,
respectively (Figures 24-25).
Based on the spectral data of compound BSB1 and comparison of its 1H and
13
C NMR with reported (Saitoh et al., 1978, Markham and Ternai, 1976) as shown in
111
Table 10, structure of compound BSB1 was identified to be isoliquiritigenin [14], first
found naturally from Dahlia variabilis (Smith and Swain, 1953).
Table 10 NMR Spectral data of compound BSB1 and isoliquiritigenin (in
DMSO-d6)
Compound BSB1
Position
*
1
H (mult., J in Hz)
Isoliquiritigenin
13
C*
1
H (mult., J in Hz)
13
C
1
-
125.8 (C)
-
125.8
2
7.74 (d, 8.7)
131.3 (CH)
7.68 (d, 8.0)
130.6
3
6.83 (d, 8.7)
115.9 (CH)
6.87 (d, 8.0)
115.8
4
-
160.3 (C)
-
159.9
5
6.83 (d, 8.7)
115.9 (CH)
6.87 (d, 8.0)
115.8
6
7.74 (d, 8.7)
131.3 (CH)
7.68 (d, 8.0)
130.6
β
7.76 (d, 16.0)
117.4 (CH)
7.82 (d, 16.0)
117.8
α
7.73 (d, 16.0)
144.3 (CH)
7.66 (d, 16.0)
143.8
β′
-
191.5 (C)
-
191.4
1′
-
112.9 (C)
-
113.2
2′
-
165.1 (C)
-
164.4
3′
6.26 (d, 2.4)
102.6 (CH)
6.33 (d, 2.5)
102.6
4′
-
165.8 (C)
-
165.4
5′
6.39 (dd, 8.8, 2.4)
108.1 (CH)
6.42 (dd, 8.0, 2.5)
107.9
6′
8.15 (d, 8.8)
132.9 (CH)
8.04 (d, 8.0)
132.3
2′-OH
13.61 (br s)
-
-
-
Carbon types were deduced from DEPT experiments.
112
1.4 Structure Determination of Compound BSB2
8
MeO
1
9
OH
7
HO 6
2α
2
10
5
3
4
O
2"
2'
3'
5'
4'
5"
O
3α
1'
6'
1"
3"
OH
OH
6"
Me
OH
4"
OMe
OH
[215]
Compound BSB2, an amorphous powder, was found to be optically active and
was analyzed for C26H34O10 from its [M+H]+ at m/z 507 in the FAB+MS (Figure 28).
Fragments at m/z 361 ([M+H]+-146) resulted from cleavage of deoxyhexose unit
without the glycosidic oxygen.
The IR spectrum of this compound showed the
presence of hydroxyl (broad band at 3401 cm-1) and aromatic (1515 cm-1) groups
(Figure 27). The UV spectrum revealed the absorption bands at 221 and 283 nm
(Figure 26).
The 1H NMR spectrum of compound BSB2 in CD3OD (Figure 29 and Table
11) showed two peaks at δ 6.10 (1H, s) and 6.59 (1H, s) due to H-5 and H-8 of the
tetrasubstituted aromatic ring, respectively, and peaks at δ 6.51 (dd, J = 8.0 and 2.0
Hz, H-6′), 6.70 (d, J = 8.0 Hz, H-5′) and 6.57 (d, J = 2.0 Hz, H-2′), ascribable to the
3H ABX system of the 3′,4′-disubstituted ring system. The peaks at δ 3.79 and δ 3.72
were attributed to the methoxy groups at C-7 and C-3′. The signal of anomeric proton
was found at δ 4.45 (d, J = 1.4 Hz) and the methyl peak characteristic of rhamnose
was observed as a doublet at δ 1.17 (J = 6.0 Hz).
Its 13C NMR data of compound BSB2 in CD3OD (Figure 30) shown in Table
11 and optical rotation are in good agreement with earlier published data (Kim et al.,
1994) which supported (+)-isolariciresinol as the aglycone moiety of compound
BSB2.
Based on the above spectral data and comparison with reported data (Kim et
al., 1994), this compound was identified as (+)-isolariciresinol-3α-O-α-Lrhamnoside [215]. The presence of this compound in this particular species is the
second report of this compound obtained as a natural products.
113
Table 11 NMR Spectral data of compound BSB2 and (+)-isolariciresinol-3α-O-αL- rhamnoside (in CD3OD)
Compound BSB2
Position
1
H (mult., J in Hz)
(+)-Isolariciresinol 3-O-α-L- rhamnoside
13
C
1
H (mult., J in Hz)
13
C
Lignan
1
2.79 (d, 8.0)
33.6
2.83 (d, 7.8)
33.6
2
1.92 (m)
40.1
2.02 (m)
40.0
3
1.88 (m)
45.5
1.86 (br t, 10.2)
45.5
4
3.80 (d, 10.2)
48.5
3.85 (d, 10.4)
48.3
5
6.10 (s)
117.1
6.16 (s)
117.1
6
-
146.1
-
146.1
7
-
149.2
-
149.2
8
6.59 (s)
112.5
6.66 (s)
112.4
9
-
128.9
-
128.9
10
-
138.1
-
138.1
1′
-
134.0
-
133.9
2′
6.57 (d, 2.0)
113.5
6.63 (d, 1.8)
113.4
3′
-
147.3
-
147.2
4′
-
145.3
-
145.2
5′
6.70 (d, 8.0)
116.1
6.75 (1H, 7.9)
116.1
6′
6.51 (dd, 8.0, 2.0)
123.2
6.59 (dd, 7.9, 1.8)
123.2
2aα
3.60-3.62 (overlapping)
65.4
3.62-3.63 (overlapping)
65.3
2bα
3.62-3.65 (overlapping)
-
3.71 (dd, 11.0, 3.7)
-
3aα
3.05 (m)
68.0
3.10 (m)
67.9
3bα
3.75-3.80 (overlapping)
-
3.80-3.82 (overlapping)
-
7-OMe
3.79 (s)
56.4
3.81 (s)
56.3
3′-OMe
3.72 (s)
56.4
3.77 (s)
56.3
1′′
4.45 (d, 1.4)
102.3
4.51 (d, 1.5)
102.3
2′′
3.82 (m)
72.3
3.84 (dd, 3.4, 1.6)
72.3
3′′
3.62 (m)
72.5
3.63 (dd, 9.3, 3.3)
72.5
4′′
3.32 (m)
73.8
3.34 (t, 9.0)
73.8
5′′
3.49 (m)
70.1
3.51 (dq, 9.0, 6.0)
70.1
6′′
1.17 (d, 6.0)
17.9
1.18 (d, 6.0)
17.9
Rhamnose
114
1.5 Structure Determination of Compound BSB3
HO
HO 4'
HO
6'
2
5' O
1' O
2'
1
3'
OH
OMe
3
4
OMe
5
6
OMe
[216]
Compound BSB3 was obtained as a white needle. Its showed a molecular ion
[M+H]
+
at m/z 347 in the FAB+MS spectrum (Figure 33), corresponding to the
molecular formula of C15H22O9. The IR spectrum showed absorption bands at 3404
(O-H stretching), 1697 (C=O stretching) and 1614 (C=C aromatic) cm-1 (Figure 32).
The UV spectrum showed the maximal absorptions at 222, 268 and 288 nm (Figure
31).
In addition, 1H and 13C NMR signals in CD3OD (Figures 34-35 and Table 12)
showed peaks assignable for an aromatic ring at δ 6.49 (2H, s, H-2, H-6)/ δ 96.1 (C-2,
C-6) and for a glucose at δ 3.29-3.48 (4H, overlapping, H-2′, 3′, 4′, 5′)/ δ 75.0 (C-2′),
78.1 (C-3′), 71.7 (C-4′), 78.4 (C-5′), δ 3.65 (1H, dd, J = 11.9, 2.4 Hz, H-6′a), δ 3.91
(1H, dd, J = 11.9, 5.2 Hz, H-6′b)/ δ 62.7 (C-6′) and δ 4.80 (1H, d, J = 7.3 Hz, H-1′)/ δ
103.2 (C-1′). Furthermore, the presence of three methoxy groups were observed at δ
3.69 (3H, s, 4-OCH3)/ δ 61.2 (4-OCH3) and δ 3.80 (6H, s, 3- OCH3, 5- OCH3)/ δ 56.6
(3- OCH3, 5- OCH3). The assignment of the methoxy groups was accomplished by
the analysis of the HMBC correlations.
Regarding the sugar unit, their directly bonded carbons were assigned by the
HMQC experiment (Figure 36). The 1H-13C long range correlations in the HMBC
spectrum (Figure 37) between anomeric proton H-1′ and C-5′ indicated a pyranose
ring with an ether linkage between C-1′ and C-5′. The presence of a diaxial-coupling
constants (J = 7.3 Hz) indicated that this sugar was a β-D-glucopyranoside. The
connection of the sugar and the aromatic ring were determined by HMBC
correlations. The sugar unit was attached at C-1 as supported by three-bond coupling
of H-1′ with C-1.
From all of the above spectroscopic data in comparison with reported values
(Shimura et al., 1988; Achenbach, Benirschike and Torrenegra, 1997), compound
115
BSB3 was identified as 3,4,5-trimethoxyphenolic-1-O-β-D-glucoside [216], first
isolated from the bark of Parabenzoin praecox (Shimura et al., 1988).
Table 12 NMR Spectral data of compound BSB3 and 3,4,5-trimethoxyphenolic1-O-β-D-glucoside (in CD3OD)
3,4,5-Trimethoxyphenolic-1-OCompound BSB3
β-D-glucoside
Position
1
H (mult., J in Hz)
13
1
C*
H (mult., J in Hz)
13
C
Aglycone
1
-
156.1 (C)
-
156.1
2
6.49 (s)
96.1 (CH)
6.48 (s)
96.2
3
-
154.8 (C)
-
154.8
4
-
134.5 (C)
-
134.5
5
-
154.8 (C)
-
154.8
6
6.49 (s)
96.1 (CH)
6.48 (s)
96.1
3, 5-OMe
3.80 (s)
56.5 (CH3)
3.80 (s)
56.6
4-OMe
3.69 (s)
61.2 (CH3)
3.69 (s)
61.2
1′
4.80 (d, 7.3)
103.2 (CH)
4.82 (d, 7.6)
103.2
2′
3.29-3.48 (m)
75.0 (CH)
3.32-3.47 (m)
75.0
3′
3.29-3.48 (m)
78.1 (CH)
3.32-3.47 (m)
78.1
4′
3.29-3.48 (m)
71.7 (CH)
3.32-3.47 (m)
71.7
5′
3.29-3.48 (m)
78.4 (CH)
3.32-3.47 (m)
78.4
6′a
3.65 (dd, 11.9, 2.4)
62.7 (CH2)
3.66 (dd, 12.3, 2.6)
62.8
6′b
3.91 (dd, 11.9, 5.2)
Glucose
*
3.92 (dd, 12.3, 5.3)
Carbon types were deduced from DEPT experiments.
116
1.6 Structure Determination of Compound BSB4
OH
3'
4' OH
2'
HO
8
7
9 O
2
1'
5'
6'
3
6
10
5
4
OH
OH
[217]
Compound BSB4 was obtained as a colorless needle and showed a molecular
ion [M-H]- in the FAB-MS spectrum at m/z 289 (Figure 40) corresponding to the
molecular formula of C15H14O6. The IR spectrum demonstrated the presence of a
hydroxyl (3404 cm-1) but no signal of a carbonyl group was observed (Figure 39).
The UV maximal absorptions at 222, 268 and 288 nm (Figure 38) were suggestive of
a flavan skeleton (Gómez et al., 1985).
The presence of a multiplet signal at δ 4.12 (1H, H-3) and two doublet of
doublet signals at δ 2.69 (H-4a) and 2.82 (H-4b) in the 1H-NMR spectrum in CD3OD
(Figure 41 and Table 13) together with the appearance of the oxygen-attached tertiary
carbon at δ 79.8 (C-2) and δ 65.1 (C-3) in the 13C NMR spectrum in CD3OD (Figure
42 and Table 13) indicated that compound BSB4 should be a flavan with oxygenation
at C-3. The protons in B-ring (H-2′, H-5′ and H-6′) formed a characteristic ABX
pattern at δ 6.94 (d, J2′,6′ = 2.1 Hz , H-2′), 6.72 (d, J5′,6′ = 8.2 Hz, H-5′) and 6.75 (dd,
J6′,5′ = 8.2 Hz and J6′,2′ = 2.1 Hz, H-6′) while the signals of H-6 and H-8 in A-ring
appeared as doublets at δ 5.91 (d, J = 2.3 Hz) and 5.88 (d, J = 2.3 Hz), respectively.
The
13
C NMR spectrum showed the methylene carbon at δ 29.2 (C-4), two methine
carbons at δ 67.4 (C-3), 79.8 (C-2), five aromatic methine at δ 95.9 (C-8), 96.4 (C-6),
115.3 (C-2′), 115.9 (C-5′), 119.4 (C-6′) and seven aromatic quarternary carbons at δ
57.3 (C-7), 100.1 (C-10), 132.2 (C-1′), 145.7 (C-3′), 145.9 (C-4′), 157.3 (C-9), 157.9
(C-5).
The 1H NMR and
13
C NMR assignments of the compound BSB4 were
performed with the aid of the DEPT method and 2D techniques such as the 1H-1H
COSY, HMQC and HMBC experiments (Figures 42-45). All protons and carbons
were assigned as shown in Table 13.
117
The absolute configuration at C-2 and C-3 of compound BSB4 has been
proved to be 2R and 3R by comparing the optical rotation value and CD spectra with
those reported in the literature (Harborne, 1982; Korver and Wilkin, 1971).
By analysis of the above spectroscopic data and comparison with previously
reported data (Agrawal, 1989), compound BSB4 was identified as (-)-epicatechin
[217], a flavan previously isolated from several plants.
118
Table 13 NMR Spectral data of compound BSB4 (in CD3OD) and (-)-epicatechin
(in DMSO-d6)
Compound BSB4
Position
1
(-)-Epicatechin
13
H (mult., J in Hz)
C*
13
C
A and C ring
2
4.76 (br s)
79.8 (CH)
78.1
3
4.12 (m)
67.4 (CH)
65.1
4a
2.69 (dd, 16.7, 2.7)
29.2 (CH2)
28.0
4b
2.82 (dd, 16.7, 4.3)
-
-
5
-
157.9 (C)
156.4
6
5.91 (d, 2.3)
96.4 (CH)
95.6
7
-
157.3 (C)
156.3
8
5.88 (d, 2.3)
95.9 (CH)
94.5
9
-
157.3 (C)
155.7
10
-
100.1 (C)
98.8
1′
-
132.2 (C)
130.7
2′
6.94 (d, 2.1)
115.3 (CH)
115.0
3′
-
145.7 (C)
144.4
4′
-
145.9 (C)
144.5
5′
6.72 (d, 8.2)
115.9 (CH)
115.0
6′
6.75 (dd, 8.2, 2.1)
119.4 (CH)
118.1
B ring
*
Carbon types were deduced from DEPT experiments.
119
1.7 Structure Determination of Compound BSB5
O
OH
1
2
6
5
3
4
OH
OH
[218]
Compound BSB5 was isolated as a colorless crystal with m.p. 194-196°C. Its
molecular formula of C7H6O4 was established by FAB+MS spectrum which showed
the [M+H]+ peak at m/z 155 (Figure 48) suggesting five degrees of unsaturation. The
IR spectrum exhibited characteristic absorption bands at 3264 cm-1 (O-H stretching),
1673 cm-1 (C=O stretching), 1601 cm-1 (C=C aromatic stretching) (Figure 47). The
UV absorption bands were found at 222, 258 and 294 nm (Figure 46).
The 1H NMR signal of compound BSB5 in CD3OD (Figure 49 and Table 14)
showed three protons belonging to 1,3,4-trisubstituted benzene ring system (ABX
system) was observed at δ 7.30 (1H, br s, H-2), 7.32 (1H, d, J = 7.5 Hz, H-6), 6.69
(1H, d, J = 7.5 Hz, H-5).
The 13C NMR signal of compound BSB5 in CD3OD (Figure 50 and Table 14)
exhibited seven signals, corresponding to three methine carbons at δ 115.7, 117.7,
123.9 and four quaternary carbons at δ 170.2, 151.5, 146.1, 123.1.
By careful analysis of the obtained spectral data and comparison of the
13
C
NMR spectral data with the previously reported data (Kaewamatawong, R., 2002) as
shown in Table 14, compound BSB5 was determined to be protocatechuic acid [218].
This compound has been isolated to be present widely in plants such as Ochna
integerrima (Kaewamatawong, R., 2002).
120
Table 14 NMR Spectral data of compound BSB5 (in CD3OD) and protocatechuic
acid (in acetone-d6)
Compound BSB5
Position
1
H (mult., J in Hz)
Protocatechuic acid
13
C
13
C
1
-
123.1
122.9
2
7.30 (br s)
123.9
123.3
3
-
146.1
145.3
4
-
151.5
150.4
5
6.69 (d, 7.5)
115.7
115.4
6
7.32 (d, 7.5)
117.7
117.2
C=O
-
170.2
167.4
121
1.8 Structure Determination of Compound BSB6
HO
HO 4'
HO
NC
6'
7
1
5' O
1' O
2'
6
3'
OH
HO
8
2
5
3
4
OH
[54]
Compound BSB6, a fine white needle, was analyzed for C14H19NO8 from its
[M+H]+ at m/z 330 and the fragment ion at m/z 168 [M+H-glucose]+ in the FAB+MS
(Figure 53). The IR spectrum showed broad adsorption at 3434 cm-1 (O-H stretching)
together with a very sharp and strong band at 2224 cm-1 (C≡N stretching) which are
expected for a conjugated nitrile group (Figure 52). The UV spectrum correlated well
with those for 1-cyanomethylene-2-cyclohexene at λmax 259 nm (Sosa et al., 1977)
(Figure 51).
The 1H NMR spectrum of compound BSB6 in D2O (Figure 54 and Table 15)
displayed three downfield signals corresponding to three olefinic protons at δ 6.23
(H-2) 6.00 (H-3) and 5.50 (H-7), respectively. The signals at δ 6.23 (H-2) and 6.00
(H-3) represent the AB part of an ABX system. The analysis of signals at δ 6.23 (H2) and 6.00 (H-3) gives the following coupling constants: J2,3 = 10.1 Hz and J3,4 = 3.0
Hz. The signal at δ 4.75 (H-6) coupled with that at 3.84 (H-5) with J5,6 = 8.2 Hz. The
signal centered at δ 4.75 (H-6) was then assigned to the allylic proton α to the Oglycosyl substituent. The low field value found for the allylic proton at δ 4.75 (H-6)
compared to 4.19 (H-4) seems to be in accordance with a stereoisomeric form in
which the nitrile group is syn with respect to the glycosidic bond. Indeed, in such a
configuration the anisotropic effect of the triple bond should deshield H-6.
The 13C NMR spectrum of compound BSB6 in D2O (Figure 55 and Table 15)
displayed a strongly deshielded olefinic carbon at δ 156.1 (C-1) and another strongly
deshielded olefinic carbon at δ 97.9 (C-7). The anomeric β-configuration of the
glucose moiety is consistent with the chemical shift noted for C-1′ as it appeared at δ
103.3. This value of anomeric carbon resembles more closely to the β-configuration
of β-D-glucopyranose (at δ 104.6) than the α configuration of the corresponding
122
epimer (at δ 100.1) (Sosa et al., 1977). The 1H NMR and
13
C spectral data of
compound BSB6 are in good agreement with earlier published data as shown in Table
15 (Sosa et al., 1977 and Wu et al., 1979). All protons and carbons were assigned by
2D NMR techniques of the HMQC and HMBC spectra (Figures 56-57).
The CD curve provides spectral information characteristic of this compound.
Compound BSB6 showed a positive maximum at 227 nm and a negative maximum at
263 nm, which are consistent with those previously reported data (Wu et al., 1979).
From all of the above spectroscopic data which are in accord with the reported
values, compound BSB6 was identified as lithospermoside [54]. This compound was
first isolated from the roots of Lithospermum purpureo-caeruleum (Sosa et al., 1977).
123
Table 15 NMR Spectral data of compound BSB6 and lithospermoside (in D2O)
Compound BSB6
Position
1
H (mult., J in Hz)
Lithospermoside
13
C*
13
C
Aglycone
1
-
156.1 (C)
157.6
2
6.23 (d, 10.1)
127.8 (CH)
129.2
3
6.00 (dd, 10.1, 3.0)
136.9 (CH)
138.7
4
4.19 (br s)
74.7 (CH)
76.2
5
3.84 (dd, 8.2, 6.1)
76.6 (CH)
78.5
6
4.75 (d, 8.2)
70.7 (CH)
72.3
7
5.50 (br d)
97.9 (CH)
99.4
8
-
118.5 (C)
120.1
1′
4.78 (d, 7.3)
103.3 (CH)
104.9
2′
3.29-3.41 (m)
73.5 (CH)
75.3
3′
3.29-3.41 (m)
76.9 (CH)
78.4
4′
3.29-3.41 (m)
70.4 (CH)
72.3
5′
3.29-3.41 (m)
76.9 (CH)
78.3
6′a
3.75 (dd, 12.3, 2.0)
61.6 (CH)
63.5
6′b
3.59 (dd, 12.3, 5.2)
-
-
Glucose
*
Carbon types were deduced from DEPT experiments.
124
1.9 Structure Determination of Compound BRC1
OH
5
O
4
10
3
6
HO 7
8
9
O
2
[219]
Compound BRC1, was obtained as a colorless crystal, having the molecular
formula of C9H6O4 which was deduced from FAB+MS spectrum and NMR spectral
data. The FAB+MS spectrum exhibited the molecular ion peak [M+H] + at m/z 179
(Figure 60). Its IR spectrum clearly revealed the presence of hydroxyl group (3003
cm-1) and carbonyl group (1646 cm-1) (Figure 59). The UV spectrum showed a
maximum absorption at 224, 250, 256, and 295 nm (Figure 58).
The 1H NMR spectrum in CD3OD of compound BRC1 (Figure 61 and Table
16) exhibited the characteristic signals due to H-2 and H-3 of a chromone skeleton at
δ 7.96 and 6.19 (1H each, d, J = 6.1 Hz), respectively. The isolated aromatic protons
with meta-coupling was observed at δ 6.20 (d, J=1.8 Hz, H-6) and δ 6.31 (d, J = 1.8
Hz, H-8). The 1H NMR spectrum in acetone-d6 of compound BRC1 (Figure 62)
showed a H-bonded phenolic proton at δ 12.74, indicating a 5-hydroxychromone
stucture.
The 13C NMR spectrum of compound BRC1 in CD3OD (Figure 63 and Table
16) displayed the resonance signals for all carbons and the multiplicity of each carbon
could assigned by the DEPT spectrum. The carbonyl carbon appeared at δ 182.5 that
was found to be similar to those commonly found for chromone (Simon et al., 1994).
Furthermore, five quarternary carbons at δ 106.5, 159.2, 163.5, 165.2, 182.5 and the
presence of four methine carbons at δ 94.7, 99.9, 111.6, 157.6 were also observed.
All protons and carbons were assigned from the HMQC and HMBC
experiments (Figures 64-65). The carbonyl carbon of chromone detected at δ 182.5
was correlated with olefinic proton at δ 7.96 (H-2) and 6.19 (H-3) with two-bond and
three-bond coupling, respectively in HMBC correlations. This clearly pointed out
that the C-2 and C-3 positions in the chromone ring should be unsubstituted.
According to the above results, and comparison of the spectral data of
compound BRC1 with those of the previously reported structure (Simon et al., 1994),
125
compound BRC1 was verified to be 5,7-dihydroxychromone [219]. This compound
was obtained previously from Calluna vulgaris (Simon et al., 1994).
Table 16 NMR spectral data of compound BRC1 and 5,7-dihydroxychromone (in
CD3OD)
Compound BRC1
Position
*
1
H (mult., J in Hz)
5,7-Dihydroxychromone
13
C*
1
H (mult., J in Hz)
13
C
2
7.96 (d, 6.1)
157.6 (CH)
7.94 (d, 6.0)
158.0
3
6.19 (d, 6.1)
111.6 (CH)
6.16 (d, 6.0)
111.7
4
-
182.5 (C)
-
183.4
5
-
163.5 (C)
-
163.5
6
6.20 (d, 1.8)
99.9 (CH)
6.17 (d, 2.1)
99.9
7
-
165.2 (C)
-
165.2
8
6.31 (d, 1.8)
94.7 (CH)
6.30 (d, 2.1)
94.7
9
-
159.2 (C)
-
159.2
10
-
106.5 (C)
-
106.5
Carbon types were deduced from DEPT experiments.
126
1.10 Structure Determination of Compound BRC2
29
28
21
11
19
HO
HO 4'
HO
1
6'
5' O
2
1' O
3
2'
3'
OH
18
20
12
13 17
8
5
26
23
24
25
27
16
9
10
4
22
14 15
7
6
[37]
Compound BRC2 was obtained as a white powder. The FAB+MS spectrum
showed molecular fragments ions at m/z 577 (Figure 67), corresponding to the
molecular formula of C35H60O6. The IR spectrum (Figure 66) exhibited an O-H
absorption at νmax 3402 cm-1 as well as C-O stretching band at 1073 cm-1 indicating the
alcohol-containing moiety of this sample.
The absorption band at 1642 cm-1
suggested the presence of C=C in the structure.
The 1H NMR spectrum in CD3OD of compound BRC2 (Figure 68 and Table
17) showed the signal in the range of δ 3.15-4.30 corresponding to a sugar moiety.
The signal at δ 4.30 was assigned to the anomeric proton (H-1′) with a coupling
constant (Jaxial, axial = 8.0 Hz) which was in agreement with a trans diaxial relationship
in β-configuration. The spectral data of aglycone part of compound BRC2 were
similar to sitosterol. Although the methyl signals of (19-CH3, 26-CH3, 27-CH3 and
29-CH3) in the range of δ 0.70-0.90 overlapped with each other, their NMR
assignments were found almost in accordance with those reported (Kojima et al.,
1990).
The
13
C NMR spectral data of compound BRC2 in CD3OD (Figure 69 and
Table 17) were almost identical with those in the literature (Kojima et al., 1990). The
comparison of the spectral data are summarized in Table 17.
On the basis of the above data by comparison of the spectral data with those
previously reported (Kojima et al., 1990), compound BRC2 was identified as
sitosteryl-3-O-β-D-glucoside [37]. This compound is the common sterol in higher
plants.
127
Table 17 NMR Spectral data of compound BRC2 (in CDCl3 + CD3OD) and
sitosteryl-3-O-β-D-glucoside (in pyridine-d5)
Compound BRC2
Position
1
H (mult., J in Hz)
sitosteryl-3-O-β-D-glucoside
13
C
13
C
Aglycone
1
0.90-1.93
37.1
37.6
2
3.15-3.50
29.0
30.3
3
0.90-1.93
79.0
78.3
4a
2.16 (m)
38.5
39.4
4b
2.29 (m)
-
-
5
-
140.1
141.0
6
5.27 (br s)
122.0
122.0
7
0.90-1.93
31.7
32.2
8
0.90-1.93
31.8
32.1
9
0.90-1.93
50.0
50.4
10
-
36.5
37.0
11
0.90-1.93
20.9
21.4
12
0.90-1.93
39.6
40.0
13
-
42.2
42.6
14
0.90-1.93
56.6
57.0
15
0.90-1.93
24.1
24.6
16
0.90-1.93
28.1
28.7
17
0.90-1.93
55.9
56.3
18
0.58 (s)
11.6
12.0
19
0.70-0.90
18.8
19.3
20
0.90-1.93
36.0
36.5
21
0.70-0.90
19.1
19.1
22
0.90-1.93
33.8
34.3
23
0.90-1.93
25.9
26.4
24
0.90-1.93
45.7
46.1
25
0.90-1.93
29.4
29.5
26
0.70-0.90
19.1
19.5
27
0.70-0.90
19.6
20.1
28
0.90-1.93
23.0
23.4
29
0.70-0.90
11.7
12.2
1′
4.30 (d, 8.0)
100.9
102.6
2′
3.15-3.50
75.5
75.4
3′
3.15-3.50
77.2
78.7
4′
3.15-3.50
70.0
71.7
5′
3.15-3.50
76.2
78.5
6′a
3.73 (dd, 11.2, 2.3)
61.7
62.9
6′b
3.60 (dd, 11.2, 4.8)
Glucose
-
128
1.11 Structure Determination of Compound BRB1
3'
HO
7
8
9 O
2
2'
1'
4' OH
5'
6'
6
10
4
5
OH
O
3
[17]
Compound BRB1 was obtained as a yellow needle with m.p. 249-251°C. The
FAB+MS showed its [M+H]+ at m/z 273
(Figure 72) suggesting the molecular
formula of C15H12O5. The UV spectrum of compound BRB1 (Figure 70) displayed
three absorption bands at 226, 288 and 332 nm. The band at 332 nm is refered to
Band I and involves the B-ring system. This band appears as a shoulder due to the
lack of conjugation between ring A and B. The bands at 288 and 226 nm are typical of
Band II which are generally considered to be due to the absorption of the A-ring
system (Markham, 1982).
The IR spectrum of compound BRB1 (Figure 71) exhibited the C=O
stretching of a conjugated carbonyl group at 1632 cm-1 which is slightly shifted to
longer wavelength due to the presence of an intramolecular hydrogen bonding
between hydroxyl aryl and keto group. The C=C stretching of aromatic ring was
observed at 1604 cm-1. The compound was clearly proved to be phenolic by the O-H
and C-O stretching bands at 3268 cm-1 and 1253 cm-1, respectively.
The 1H NMR spectrum in CD3OD of compound BRB1 (Figure 73 and Table
18) was characteristic of as a flavanone. Protons in the B-ring (H-2′, H-6′ and H-3′,
H-5′) formed a characteristic AA′BB′ pattern at δ 7.30 (d, J2′,3′ = J6′,5′ = 8.6 Hz, H-2′
and H-6′) and 6.80 (d, J3′,2′ = J5′,6′ = 8.6 Hz, H-3′ and H-5′), while the signals of H-6
and H-8 in the A-ring appeared as a doublet at δ 5.85 (d, J = 2.0 Hz) and 5.86 (d, J =
2.0 Hz), respectively. A doublet of doublet of H-2 indicated the cis-relationship
between H-2 and H-3a with J2,3a = 3.0 Hz and trans-relationship between H-2 and H3b with (J2,3b = 12.9 Hz). The signals at δ 2.68 (dd, J3a,3b = 16.2 Hz and J3a,2 = 3.0 Hz)
and 3.15 (dd, J3b,3a = 16.2 Hz and J3b,2 = 12.9 Hz) were referred to H-3a and H-3b,
respectively.
129
The 13C NMR spectrum of compound BRB1 in CD3OD (Figure 74 and Table
18) showed 15 signals for 15 carbon atoms. The types of carbons are classified by the
analysis of the DEPT spectrum as shown in Table. The 13C NMR spectral data were
in close agreement with the previously published values (Agrawal, 1989) as shown in
Table. In order to confirm the chemical shifts of protons and carbons of compound
BRB1, the HMQC and HMBC experiments were performed (Figure 75-76).
Compound BRB1 was optically active with the optical rotation of [α] D23 -13°
(MeOH, c 0.23). The absolute configuration at C-2 of this compound has been proved
to be in an S-configuration by comparison of the optical rotation value with those
reported in the literature (Hsieh, Fang and Cheng, 1998).
Based on the above data, compound BRB1 was identified as (2S)-naringenin
[17]. This compound was obtained previously from Artemisia dracunculus (Balza
and Tower, 1984).
130
Table 18 NMR Spectral data of compound BRB1 (in CD3OD) and
(2S)-naringenin (in acetone-d6)
Compound BRB3
Position
1
(2S)-Naringenin
13
H (mult., J in Hz)
C*
13
C
A and C ring
2
5.30 (dd, 12.9, 3.0)
80.5 (CH)
80.1
3a
2.68 (dd, 16.2, 3.0)
44.1 (CH2)
43.7
3b
3.15 (dd, 16.2, 12.9
-
4
-
197.8 (C)
197.3
5
-
165.5 (C)
165.0
6
5.85 (d, 2.0)
97.1 (CH)
97.0
7
-
168.5 (C)
168.0
8
5.86 (d, 2.0)
96.2 (CH)
96.1
9
-
164.9 (C)
164.5
10
-
103.3 (C)
103.1
131.1 (C)
130.7
B ring
1′
*
2′
7.30 (d, 8.6)
129.0 (CH)
128.8
3′
6.80 (d, 8.6)
116.3 (CH)
116.2
4′
-
159.1 (C)
158.5
5′
6.80 (d, 8.6)
116.3 (CH)
116.2
6′
7.30 (d, 8.6)
129.0 (CH)
128.8
Carbon types were deduced from DEPT experiments.
131
1.12 Structure Determination of Compound BRB2
OH
3'
8
HO
9 O
2'
2 1'
7
6
4'
OH
5'
6'
10 4
5
OH
O
3
[220]
Compound BRB2 was obtained as a yellow needle with m.p. 325-328°C. The
UV spectrum displayed absorption bands at 221, 255, 267 and 350 nm (Figure 77).
The IR spectrum exhibited absorption bands at 3395 (O-H stretching), 1657 (C=O
stretching) and 1608 (C=C aromatic ring) cm-1 (Figure 78). The FAB+MS showed its
[M+H] + at m/z 287 (Figure) suggesting the molecular formula of C15H10O6 (Figure
79).
The 1H NMR spectrum in DMSO-d6 of compound BRB2 (Figure 80 and Table
19) showed a H-bonded phenolic proton at δ 12.97 ppm, indicating a 5hydroxyflavone structure. The protons in B-ring (H-2′, H-5′ and H-6′) formed a
characteristic ABX pattern at δ 7.39 (d, J2′,6′ = 2.0 Hz , H-2′), 6.87 (d, J5′,6′ = 8.5 Hz,
H-5′) and 7.41 (dd, J6′,5′ = 8.5 Hz and J6′,2′ = 2.0 Hz, H-6′) while the signals of H-6
and H-8 in A-ring appeared as a doublet at δ 6.17 (d, J = 2.0 Hz) and 6.43 (d, J = 2.0
Hz), respectively. An olefinic singlet proton at δ 6.66 was assigned to H-3 by its
HMBC correlations with C-10 (103.7) and C-1′ (121.5).
The
13
C NMR spectrum of compound BRB2 in DMSO-d6 (Figure 81 and
Table 19) showed 15 signals for 15 carbon atoms. The types of carbons are classified
by the analysis of the DEPT spectrum as shown in Table 19.
Based on the above spectral evidence, and comparison of the spectral data of
compound BRB2 with those previously reported (Agrawal, 1989), together with the
information from the HMBC and HMQC experiments (Figures 82-83), compound
BRB2 was identified as luteolin [220]. This compound occurred in many plants of
the
family
Leguminosae,
Resedaceae,
Euphorbiaceae,
Umbelliferae,
Scrophulariaceae, Fabaceae, Asteraceae, Cistaceae, Passifloraceae, Yerbenaceae and
Hepaticae (Buckingham, 2001).
132
Table 19 NMR Spectral data of compound BRB2 and luteolin (in DMSO-d6)
Compound BRB2
Position
1
Luteolin
13
H (mult., J in Hz)
C*
13
C
A and C ring
2
-
163.9 (C)
164.5
3
6.66 (s)
102.8 (CH)
103.3
4
-
181.6(C)
182.2
5
-
161.5 (C)
162.1
6
6.17 (d, 2.0)
98.8 (CH)
99.2
7
-
164.1 (C)
164.7
8
6.43 (d, 2.0)
93.8 (CH)
94.2
9
-
157.3 (C)
157.9
10
-
103.7 (C)
104.2
5-OH
12.97 (s)
-
-
1′
-
121.5 (C)
122.1
2′
7.39 (d, 2.0)
113.3 (CH)
113.8
3′
-
145.7 (C)
146.2
4′
-
149.7 (C)
150.2
5′
6.87 (d, 8.5)
116.0 (CH)
116.4
6′
7.41 (dd, 8.5, 2.0)
119.0 (CH)
119.3
B ring
*
Carbon types were deduced from DEPT experiments.
133
1.13 Structure Determination of Compound BRB3
OH
3'
2'
8
HO
9 O 2 1'
7
6
4' OH
5'
6'
3
4
5 10
OH
O
[16]
Compound BRB3 was obtained as a pale yellow needle with m.p. 198-200°C,
showed its [M+H] + at m/z 289 in FAB+MS spectrum (Figure 86) corresponding to the
molecular formula of C15H12O6.
The IR spectrum showed absorption bands at
-1
3366 cm (O-H stretching), and 1632 cm-1 (C=O stretching) cm-1 (Figure 85). The
UV absorptions at 224, 288 and 328 nm (Figure 84) were indicative of a flavanone
skeleton (Markham, 1982).
The 1H-NMR spectrum of compound BRB3 in CD3OD (Figure 87 and Table
20) revealed a doublet of doublet of H-2, indicated the cis-relationship between H-2
and H-3a (J2,3a = 2.8 Hz) and trans-relationship between H-2 and H-3b (J2,3b = 12.6
Hz). The A-ring showed an AB coupling system of the two aromatic protons at H-6
and H-8. The B-ring exhibited signals for an ABX pattern at δ 6.91 (d, J2′,6′ = 2.2 Hz
, H-2′), 6.76 (d, J5′,6′ = 8.0 Hz, H-5′) and 6.78 (dd, J6′,5′ = 8.0 Hz and J6′,2′ = 2.2 Hz,
H-6′). The 13C NMR spectrum of compound BRB3 in CD3OD (Figure 88 and Table
20) showed 15 signals for 15 carbon atoms. The types of carbons are classified by the
analysis of the DEPT spectrum as shown in Table. Its
13
C NMR data are in good
agreement with earlier published data (Agrawal, 1989). The successful assignments
of compound BRB3 were accomplished by application of 2D NMR, including the
HMQC and HMBC experiments (Figures 89-90).
The absolute configuration at C-2 of compound BRB3 has been proved to be
S-configuration by comparing the optical rotation value with those reported in the
literature (Harborne and Mabry, 1982). On the basis of the above spectroscopic data,
this compound was identified as (2S)-eriodictyol [16].
previously separated from several plants.
This compound was
134
Table 20 NMR Spectral data of compound BRB3 (in CD3OD) and
(2S)-eriodictyol (in DMSO-d6)
Compound BRB3
Position
1
(2S)-Eriodictyol
13
H (mult., J in Hz)
C*
13
C
A and C ring
2
5.25 (dd, 12.6, 2.8)
80.5 (CH)
78.3
3a
2.63 (dd, 17.0, 2.8)
44.1 (CH2)
42.2
3b
3.05 (dd, 17.0, 12.6)
-
-
4
-
197.7 (C)
196.2
5
-
165.5 (C)
163.4
6
5.87 (d, 2.4)
97.0 (CH)
95.7
7
-
168.4 (C)
166.6
8
5.91 (d, 2.4)
96.2 (CH)
94.8
9
-
164.8 (C)
162.8
10
-
103.3 (C)
101.7
1′
-
131.8 (C)
129.4
2′
6.91 (d, 2.2)
114.7 (CH)
114.2
3′
-
146.5 (C)
145.1
4′
-
146.9 (C)
145.6
5′
6.76 (d, 8.0)
116.2 (CH)
115.3
6′
6.78 (dd, 8.0, 2.2)
119.2 (CH)
117.8
B ring
*
Carbon types were deduced from DEPT experiments.
135
1.14 Structure Determination of Compound BRB4
OH
3'
4' OH
2'
8
HO
9 O 2 1'
7
6
5'
6'
3
5 10 4
OH
O
OH
[221]
Compound BRB4 was obtained as a pale yellow needle with m.p. 238-241°C.
A molecular formula of C15H12O7 was established based on the FAB+MS which
exhibited [M+H]+ at m/z 305
(Figure 93).
The UV spectrum displayed three
absorption bands at 222, 290 and 325 nm (Figure 91). The IR spectrum indicated the
presence of hydroxy (3412 cm-1) and carbonyl (1639 cm-1) groups (Figure 92).
The 1H NMR spectrum in CD3OD of compound BRB4 (Figure 94 and Table
21) showed the typical AB-coupled protons at δ 4.85 and 4.49 (J = 14.0 Hz, 1H each)
due to H-2 and H-3 of a dihydroflavonol, respectively. By comparison of 1H NMR
spectral data of compound BRB4 with those of compounds BRB2 and BRB3, similar
coupling patterns of protons as an AB pattern at ring A and ABX pattern at ring C
could be observed. The
13
C NMR spectrum of compound BRB4 in CD3OD (Figure
95 and Table 21) showed 15 signals for 15 carbon atoms, corresponding to a
dihydroflavonol. The 1H and 13C NMR assignments were performed using the DEPT,
HMQC and HMBC experiments (Figures 96-97). Thus, compound BRB4 possessed
the 5,7,3′,4′-tetrahydroxy dihydroflavonol skeleton.
The absolute configuration of compound BRB4 is (2R, 3R). The CD spectra
and optical rotation of compound BRB4 are in good agreement with those previously
published (Lundgren and Theander, 1988).
Compound BRB4 was identified as (+)-taxifolin (trans-dihydroquercetin)
[221] based on the above spectral data. The 1H and
13
C NMR spectra were in close
agreement with previously published values (Lundgren and Theander, 1988) as shown
in Table 21. This compound has been isolated from Pinus sylvestris (Lundgren and
Theander, 1988).
136
Table 21 NMR Spectral data of compound BRB4 and (+)-taxifolin (in CD3OD)
Compound BRB4
Position
1
H (mult., J in Hz)
13
(+)-Taxifolin
C*
1
H (mult., J in Hz)
13
C
A and C ring
2
4.85 (d, 14.0)
85.1 (CH)
4.92 (d, 11.3)
84.7
3
4.49 (d, 14.0)
73.6 (CH)
4.49 (d, 11.3)
73.2
4
-
198.4 (C)
-
197.9
5
-
165.3 (C)
-
164.7
6
5.85 (d, 2.5)
97.3 (CH)
5.89 (d, 2.2)
97.0
7
-
168.8 (C)
-
168.1
8
5.92 (d, 2.5)
96.3 (CH)
5.93 (d, 2.2)
95.6
9
-
164.5 (C)
-
164.0
10
-
101.8 (C)
-
101.3
1′
-
129.9 (C)
-
129.3
2′
6.91 (d, 2.0)
115.9 (CH)
6.97 (d, 2.0)
115.6
3′
-
146.3 (C)
-
145.8
4′
-
147.1 (C)
-
146.5
5′
6.78 (d, 8.0)
116.0 (CH)
6.81 (d, 8.2)
115.6
6′
6.82 (dd, 8.0, 2.0)
120.9 (CH)
6.85 (dd, 8.2, 2.0)
120.4
B ring
*
Carbon types were deduced from DEPT experiments.
137
1.15 Structure Determination of Compound BRB5
8
MeO
9
1
2α
2
OH
7
HO 6
5 10
OMe
3
4
5"
O
2"
2'
3'
4'
1"
3α
1'
6'
MeO 5'
O
3"
OH
OH
6"
Me
OH
4"
OMe
OH
[222]
Compound BRB5, an amorphous solid, was found to be optically active and
was analyzed for C28H38O12 from its [M+K]+ at m/z 605 in the FAB+MS spectrum
(Figure 100).
The IR spectrum of compound BRB5 exhibited characteristic
absorption bands at 3402 cm-1 (O-H stretching) and 1614 cm-1 (C=C aromatic) (Figure
99). The UV spectrum showed the absorption bands at 221 and 278 nm (Figure 98).
The 1H NMR spectrum of compound BRB5 in CD3OD (Figure 101 and Table
22) showed the presence of two singlet peaks at δ 6.50 and 6.25 belonging to the H-8
and H-2′ of the aromatic rings. The peaks at δ 3.15, 3.64 and 3.76 were attributed to
the methoxy groups at C-5, C-3′ and C-5′, C-7, respectively, as indicated by HMBC
spectra. The signal of anomeric proton was found at δ 4.60 (d, J = 1.2 Hz) and the
methyl peak characteristic of rhamnose was observed as a doublet at δ 1.19 (J = 6.1
Hz).
The
13
C NMR data in CD3OD (Figure 102 and Table 22) and DEPT
experiment showed 28 carbon signals, corresponding to four methoxyl carbons, one
methyl carbon, three methylene carbons, eleven methine carbons and nine quarternary
carbons (Figure). In addition, six peaks at δ 18.6, 70.2, 72.5, 73.1, 74.1 and 102.1
were assigned to C-1′′ to C-6′′ of α-L-rhamnosyl moiety. The location of glycosidic
linkage was elucidated by the analysis of 2D NMR spectra, especially the 1H-1H
COSY, HMQC and HMBC spectra (Figures 103-105).
By analysis of the above spectroscopic data and comparison of its
13
C NMR
and optical rotation values with previously reported data (Fuchino et al., 1995),
compound BRB5 was thus identified as (+)-lyoniresinol-3α-O-α-L-rhamnoside [222].
This compound has been reported from Ulmus thomasii (Hostettler and Seikel, 1969).
138
Table 22 NMR Spectral data of compound BRB5 (in CD3OD) and
(+)-lyoniresinol-3α-O-α-L-rhamnoside (in pyridine-d5)
(+)-Lyoniresinol 3α-O-α-L-
Compound BRB5
Position
rhamnoside
1
H (mult., J in Hz)
13
C
13
C
Lignan
1a
2.50 (dd, 15.0, 11.9)
33.7 (CH2)
33.6
1b
2.65 (dd, 15.0, 4.6)
-
-
2
1.55 (m)
41.0 (CH)
41.3
3
2.00 (m)
46.5 (CH)
46.0
4
4.21 (d, 5.8)
43.0 (CH)
42.4
5
-
147.5 (C)
147.9
6
-
138.9 (C)
139.4
7
-
148.7 (C)
148.4
8
6.50 (s)
107.8 (CH)
107.5
9
-
130.1 (C)
129.7
10
-
126.0 (C)
126.1
1′
-
139.2 (C)
138.6
2′
6.25 (s)
106.7 (CH)
107.1
3′
-
149.1 (C)
149.0
4′
-
134.7 (C)
135.7
5′
-
149.1 (C)
149.0
6′
6.25 (s)
106.7 (CH)
107.1
2aα
3.38 (dd, 10.8, 7.2)
66.3 (CH2)
65.6
2bα
3.52-3.54 (overlapping)
-
-
3aα
3.23 (m)
69.7 (CH2)
69.7
3bα
3.50-3.55 (overlapping)
-
-
5-OMe
3.15 (s)
60.1 (CH3)
59.8
7-OMe
3.76 (s)
56.6 (CH3)
56.1
3′, 5′-OMe
3.64 (s)
56.8 (CH3)
56.5
1′′
4.60 (d, 1.2)
102.0 (CH)
102.1
2′′
3.79 (dd, 3.0, 1.8))
72.4 (CH)
72.5
3′′
3.60 (dd, 9.5, 3.6)
72.6 (CH)
73.1
4′′
3.27 (t, 9.4)
73.9 (CH)
74.1
5′′
3.45 (dq, 9.4, 6.1)
70.1 (CH)
70.2
6′′
1.19 (d, 6.1)
17.9 (CH3)
18.6
Rhamnose
139
1.16 Structure Determination of Compound BRB6
HO
HO 4'
HO
OH
5
6'
O
4
6
5' O
1' O
2'
3'
OH
3
7
8
O
2
[223]
Compound BRB6 was obtained as a white powder by crystallization from
methanol. The FAB+MS spectrum showed molecular fragments ions at m/z 341
(Figure 107), corresponding to C15H16O9. The UV spectrum showed absorption bands
at 221, 252, 256 and 288 nm (Figure 106).
By using the data of 1H and 13C NMR in CD3OD of compound BRB6 (Figures
108-109 and Table 23), the existence of chromone unit and the glucose unit were
established. From
13
C NMR spectrum, the sugar moiety showed signals at δ 62.4,
71.2, 74.7, 77.8, 78.4, 101.6 and the chromone unit exhibited signals at δ 96.2, 101.3,
108.4, 112.0, 158.6, 159.5, 163.4, 164.9, 183.6. The mode of glucosidic linkage was
determined to be in β-configuration based on the coupling constant of the anomeric
proton signal at δ 4.98 (1H, d, J = 7.0 Hz).
Based on the comparison of its 1H and
13
C NMR data with those reported
previously data (Simon et al., 1994), compound BRB6 was identified as
5-hydroxychromone-7-β-D-glucoside [223].
Calluna vulgaris (Simon et al., 1994).
This compound has been found in
140
Table 23 NMR spectral data of compound BRB6 and 5-hydroxychromone-7-βD-glucoside (in CD3OD)
5-Hydroxychromone-7-β-D-
Compound BRB6
Position
glucoside
1
H (mult., J in Hz)
13
C
1
H (mult., J in Hz)
13
C
Chromone
2
7.96 (d, 6.1)
158.6
7.94 (d, 6.0)
158.0
3
6.19 (d, 6.1)
112.0
6.16 (d, 6.0)
111.7
4
-
183.6
-
183.4
5
-
163.4
-
163.5
6
6.20 (d, 2.1)
101.3
6.17 (d, 2.1)
99.9
7
-
164.9
-
165.2
8
6.31 (d, 2.1)
96.2
6.30 (d, 2.1)
94.7
9
-
159.5
-
159.2
10
-
108.4
-
106.5
1′
4.98 (d, 7.0)
101.6
5.00 (d, 7.0)
101.6
2′
3.35-3.45 (m)
74.7
3.35-3.50 (m)
74.7
3′
3.35-3.45 (m)
77.8
3.35-3.50 (m)
77.9
4′
3.35-3.45 (m)
71.2
3.35-3.50 (m)
71.2
5′
3.35-3.45 (m)
78.4
3.35-3.50 (m)
78.4
6′a
3.81 (dd, 12.0, 2.0)
62.4
3.87 (dd, 12.1, 1.9)
62.4
6′b
3.62 (dd, 12.0, 5.5)
-
3.67 (dd, 12.1, 5.6)
-
Glucose
141
1.17 Structure Determination of Compound BRB7
HO
HO 4'
HO
NC
6'
5' O
1' O
2'
6
3'
OH
5
8
7
1
2
3
4
OH
[224]
Compound BRB7, a white powder, showed a typical strong nitrile absorption
(C≡N stretching) at 2220 cm-1 together with a C=C stretching vibration at 1620 cm-1
in the IR spectrum (Figure 111) and also gave an absorption maximum at 258 nm in
the UV spectrum (Figure 110), suggesting of the presence of an α, β, γ, δ-unsaturated
nitrile group in the molecule (Nakanishi, K. et al., 1978). The FAB+MS afforded the
[M+H]+ peak at m/z 314 and an intense fragment ion at m/z 152 ([M+H]+-162
[glucose unit]), indicating that compound BRB7 is a monoglycoside and carries a
glucose as a sugar unit (Figure 112).
The 1H NMR and 13C NMR of compound BRB7 in CD3OD (Figures 113-114
and Table 24) differed from those of compound BSB6 at position 5. From 1H NMR
of compound BRB7, the doublet of doublet of doublet signals at δ 1.92 (J5a,5b = 13.7,
J5a,6 = 7.8 and J5a,4 = 6.4 Hz) and 2.16 (J5b,5a = 13.7, J5b,4 = 5.0 and J5b,6 = 3.1 Hz)
were assigned as H-5a and H-5b, respectively. Detailed 1H NMR and
13
C NMR
assignments of the compound BRB7 were performed with the aid of the DEPT
method and 2D techniques such as the 1H-1H COSY, HMQC and HMBC experiments
(Figures 115-117) and all protons and carbons were assigned as shown in Table 24.
The location of the β–D-glucopyranosyl residue on the aglycone was then determined.
The sugar moiety exhibited signals at δ 63.2, 71.8, 74.5, 78.0, 78.2 and 101.6. The
observed vicinal coupling constants of J = 7.3 Hz between the trans diaxial
oxymethine protons H-1′ and H-2′ suggested that H-1′ were β-anomeric protons. The
stereochemistry for this compound was assigned based on the comparison of the
optical rotation with reported data (Nakanishi et al., 1994).
Thus, compound BRB7 was identified as menisdaurin [224], a cyanoglucoside
previously isolated from Purshia tridentata (Nakanishi et al., 1994).
142
Table 24 NMR Spectral data of compound BRB7 and menisdaurin (in CD3OD)
Compound BRB7
Position
1
H (mult., J in Hz)
Menisdaurin
13
*
1
C
H (mult., J in Hz)
13
C
Aglycone
1
-
157.1 (C)
-
157.8
2
6.18 (d, 10.1)
127.8 (CH)
6.29 (ddd, 10.0, 1.5, 0.8)
128.4
3
6.10 (dd, 10.1, 3.6)
140.6 (CH)
6.21 (ddd, 10.0, 3.5, 0.8)
141.3
4
4.26 (br s)
65.4 (CH)
4.36 (dddd, 6.3, 5.5, 3.5, 1.5)
66.0
5a
1.92 (ddd, 13.7, 7.8, 6.4)
36.1 (CH2)
2.04 (ddd, 13.2, 8.0, 6.3)
36.7
5b
2.16 (ddd, 13.7, 5.0, 3.1)
-
2.25 (ddd, 13.2, 5.5, 3.5)
-
6
4.82 (br s)
72.5 (CH)
4.93 (ddd, 8.0, 3.5, 1.3)
73.2
7
5.41 (br d)
96.8 (CH)
5.50 (ddd, 0.3, 0.8, 1.3)
97.6
8
-
118.0 (C)
-
118.7
1′
4.45 (d, 7.3)
101.6 (CH)
4.55 (ddd, 8.0, 3.5, 1.3)
102.3
2′
3.29-3.40 (m)
74.5 (CH)
3.34 (dd, 9.0, 7.5)
75.2
3′
3.29-3.40 (m)
78.0 (CH)
3.39 (dd, 9.0, 9.0)
78.7
4′
3.29-3.40 (m)
71.8 (CH)
3.29 (dd, 9.0, 9.0)
72.4
5′
3.29-3.40 (m)
78.2 (CH)
3.34 (ddd, 9.0, 6.2, 2.2)
78.8
6′a
3.56 (dd, 12.3, 6.1)
63.2 (CH)
3.67 (dd, 11.8, 6.2)
63.8
6′b
3.79 (dd, 12.3, 2.1)
-
3.89 (dd, 11.8, 2.2)
-
Glucose
*
Carbon types were deduced from DEPT experiments.
143
1.18 Structure Determination of Compound CBE1
18
13
15
14
16
17
12
11
10
9
8
7 6
O
4
5
3
2
1
[225]
Compound CBE1 was obtained as a colorless oil. Its molecular formula of
C18H30O was deduced from [M]+ ion of the EIMS spectrum at m/z 262 (Figure 119)
suggesting four degrees of unsaturation. The IR spectrum exhibited characteristic
absorption bands at 3019 cm-1 (C-H stretching) and 1712 cm-1 (C=O stretching)
(Figure 118).
The 1H NMR spectrum in CDCl3 of compound CBE1 (Figure 120 and Table
25) revealed the methyl protons at δ 1.59 (H-17), 1.60 (H-18), 1.62 (H-16), 1.68 (H15) and 2.14 (H-1), methylene protons at δ 1.96-2.45 and olefinic protons at δ 5.085.09.
The 13C NMR spectrum of compound CBE1 in CDCl3 (Figure 121 and Table
25) showed the carbonyl carbon of the ketone at δ 208.8 (C-2) and the olefinic
carbons at δ 122.5 (C-5), 124.0 (C-9), 124.4 (C-13), 131.2 (C-14), 134.9 (C-10), and
136.2 (C-6). The carbonyl group could be placed at C-2 according to the HMBC
correlations of C-2 with H-1, H-3 (two-bond correlation) and H-4 (three-bond
correlation).
The olefinic carbons, C-5, C-9 and C-13 showed long-range (3J)
coupling with the methyl protons, H-16, H-17 and H-15, respectively. The types of
carbons are classified by the analysis of the DEPT experiment as shown in Table. All
protons and carbons were assigned by analysis of the 1H-1H COSY, HMQC and
HMBC spectra (Figures 122-124).
Compound CBE1 was identified as farnesyl acetone [225] according to the
above spectral data, which as confirmed by comparing them with the previously
published data (Ravi et al., 1982). This compound was commonly found in plants
and also found in the brown alga Cystophora moniliformis (Ravi et al., 1982).
144
Table 25 NMR Spectral data of compound CBE1 and farnesyl acetone
(in CDCl3)
Compound CBE1
Position
*
1
H (mult., J in Hz)
Farnesyl acetone
13
C*
13
C
1
2.14 (s)
29.9 (CH3)
29.8
2
-
208.8 (C)
208.4
3
2.45 (t, 7.5)
43.8 (CH2)
43.7
4
2.26 (q, 7.3)
22.5 (CH2)
22.5
5
5.08 (m)
122.5 (CH)
122.5
6
-
136.4 (C)
136.2
7
1.96 (m)
39.6 (CH2)
39.7
8
1.98 (m)
26.7 (CH2)
26.7
9
5.08 (m)
124.0 (CH)
124.3
10
-
135.0 (C)
134.9
11
2.07 (m)
39.7 (CH2)
39.7
12
2.06 (m)
26.5 (CH2)
26.5
13
5.09 (m)
124.4 (CH)
124.3
14
-
131.2 (C)
131.0
15
1.68 (s)
25.7 (CH3)
25.7
16
1.62 (s)
16.0 (CH3)
16.0
17
1.59 (s)
16.0 (CH3)
16.0
18
1.60 (s)
17.7 (CH3)
17.7
Carbon types were deduced from DEPT experiments.
145
1.19 Structure Determination of Compound CBE2
18
5
4
3
6
7
2
16
1
19
8
9
10
11
15
13 14
12
17
O 20 OH
[163]
Compound CBE2 was obtained as a colorless oil. Its molecular formula of
C20H30O2 was established by EIMS with the [M]+ peak at m/z 302, suggesting five
degrees of unsaturation (Figure 127).
The IR spectrum exhibited characteristic
absorption bands at 3445 cm-1 (O-H stretching) and 1699 cm-1 (C=O stretching)
(Figure 126). The UV spectrum exhibited absorption bands at 230 nm (Figure 125).
The 1H NMR spectrum in CDCl3 of compound CBE2 (Figure 128 and Table
26) showed the presence of two almost overlapped doublets at δ 0.80 (J = 6.8 Hz, H17) and 0.83 (J = 6.7 Hz, H-16) together with multiplet at δ 1.48 (H-15)
corresponding to non-equivalent methyl protons in an isopropyl group which is
probably bonded to an asymmetric carbon. Two singlets of three protons each at δ
1.65 (H-19) and 1.81 (H-18) corresponded to two methyl groups bonded to olefinic
carbons. Multiplets between δ 1.35 to 3.08 corresponded to methylene and methine
protons. The two mutually coupled trans-olefenic protons at δ 5.21 (dd, J2,1 = 9.8 and
J2,3 = 15.6 Hz, H-2) and 6.07 (d, J = 15.6 Hz, H-3) and three olefenic protons at δ 5.19
(H-7), 5.58 (H-5) and δ 6.05 (H-11) could be detected. The downfield one proton at δ
6.07 (H-11) was assigned to be the β-proton of an α, β-unsaturated carbonyl group
(-CH=C-CO).
The 13C NMR spectrum of compound CBE2 in CDCl3 (Figure 129 and Table
26) revealed the presence of 20 carbons consisting of four methyl, five methylene,
seven methine and four quarternary carbons from the DEPT experiment. Among the
quarternary carbons, one was the carboxyl carbon (C=O). The presence of 20 carbons
led to a conclusion that compound CBE2 was a diterpene. Considering the main
skeletons of all diterpenes summarized in literatures (Devon and Scott, 1972), only
cembrane and abietane-type diterpenes posses an isopropyl group.
These two
146
skeletons were therefore taken into consideration. Further literature reviews showed
that several cembrane-type diterpenes were found as constituents in some Croton
species (Roengsumran et al., 1998) and none of the abietane-type diterpenes were
reported so far.
The 2D NMR technique, 1H-1H COSY (Figure 130) clearly showed coupling
between signals at δ 0.80 (H-17) and 0.83 (H-16) and multiplet of a methine proton at
δ 1.48 (H-15). This evidence suggested the presence of an isopropyl group. The
methyl signals at δ 1.65 (H-19) and 1.81 (H-18) corresponding to methyl groups
bonded to olefinic carbons, gave a correlation to olefinic protons at δ 5.19 (H-7) and
5.58 (H-5), respectively. From the above evidence together with the HMBC and
HMQC spectra (Figures 132-133) the presence of the two methyl groups could
therefore be placed at C-4 and C-8. In addition to the correlation between signals at δ
6.05 (H-11), which was assigned to the β-proton of an α,β-unsaturated carbonyl
moiety, showed cross-peak with multiplet at δ 1.99 (H-13) in HMBC spectral data
was observed. The above data led to place double bonds at C-2/ C-3, C-4/ C-5, C-7/
C-8 and C-11/ C-12 in a cembrane skeleton. The trans-double bond was assigned at
C-2/ C-3. The double bond located at C-11/ C-12 implying that C-20 of the cembrane
skeleton should be a carboxyl group. After the placement of double bond locations, it
was therefore possible to assign all correlated protons as shown in Table 26.
The NOESY experiment (Figure 6 and 131) was performed between H-2, H-5/
H-18, H-2/ H-16, H-3/ H-19, H-7/ H-9, H-11/ H-13 and H-14/ H-17 were observed.
The NOESY experiment of compound CBE2 was suggested the configuration of all
double bonds. The stereochemistry for this compound was established based on the
comparing of the optical rotation with the reported data (Sato et al., 1991).
The structure of compound CBE2 were proposed to be poilaneic acid [163],
based on the above spectral evidence and reported data (Sato et al., 1991). This
compound was previously found in Croton poilanei (Sato et al., 1991).
147
18
5
7
4
3 2
6
16
1
8
19
9
10
11
13
12
14
15
17
O 20 OH
Figure 6 NOESY experiment in compound CBE2
148
Table 26 NMR Spectral data of compound CBE2 and poilaneic acid (in CDCl3)
Compound CBE2
Position
*
1
H (mult., J in Hz)
Poilaneic acid
13
C*
1
H (mult., J in Hz)
13
C
1
1.73 (m)
47.9 (CH)
1.5-2.5 (m)
48.0
2
5.21 (dd, 9.8, 15.6)
131.3 (CH)
5.21 (dd, 9.5, 15.5)
131.3
3
6.07 (d, 15.6)
131.0 (CH)
6.05 (d, 15.5)
131.0
4
-
135.1 (C)
-
135.2
5
5.58 (br t)
125.7 (CH)
5.56 (dd, 6.0, 9.5)
125.7
6a
3.08 (m)
26.2 (CH2)
3.05 (ddd, 6.0, 9.5, 15.5)
26.3
6b
2.45 (m)
-
1.5-2.5 (m)
-
7
5.19 (br d)
127.9 (CH)
5.21 (dd, 6.0, 9.5)
128.0
8
-
131.3 (C)
-
131.3
9a
2.28 (m)
38.5 (CH2)
1.5-2.5 (m)
38.6
9b
2.01 (m)
-
1.5-2.5 (m)
-
10a
2.91 (m)
25.8 (CH2)
1.5-2.5 (m)
25.9
10b
2.49 (m)
-
1.5-2.5 (m)
-
11
6.05 (dd, 5.0, 6.7)
147.8 (CH)
6.05 (dd, 4.5, 6.5)
147.8
12
-
128.7 (C)
-
128.9
13a
2.52 (m)
32.1 (CH2)
1.5-2.5 (m)
32.2
13b
1.99 (m)
-
1.5-2.5 (m)
-
14a
1.78 (m)
29.4 (CH2)
1.5-2.5 (m)
29.5
14b
1.35 (m)
-
1.5-2.5 (m)
-
15
1.48 (m)
32.7 (CH)
1.5-2.5 (m)
32.8
16
0.83 (d, 6.7)
21.0 (CH3)
0.83 (d, 6.5)
20.9
17
0.80 (d, 6.8)
19.3 (CH3)
0.80 (d, 6.5)
19.4
18
1.81 (br s)
20.0 (CH3)
1.82 (t, 1.5)
19.9
19
1.65 (br s)
14.5 (CH3)
1.66 (t, 1.5)
14.5
20
-
173.1 (C)
-
173.7
Carbon types were deduced from DEPT experiments.
149
1.20 Structure Determination of Compound CBE3
O
2
3
HO 4
1
H
6
5
[226]
Compound CBE3 was a colorless needle with m.p. 195-198°C. Its molecular
formula of C7H6O2 was established by EIMS which showed the [M]+ peak at m/z 122,
suggesting five degrees of unsaturation (Figure 136). The IR spectrum exhibited
characteristic absorption bands at 3164 cm-1 (O-H stretching), 1666 cm-1 (C=O
stretching), 1597 cm-1 (C=C stretching aromatic) (Figure 133). The UV spectrum
exhibited absorption bands at 222 and 284 nm (Figure 132).
The 1H NMR spectrum in CDCl3 of compound CBE3 (Figure 137 and Table
27) showed the protons on the aromatic ring. The protons on the aromatic ring (H-2,
H-6 and H-3, H-5) formed a characteristic AA′BB′ pattern at δ 7.82 (d, J2,3 = J6,5 = 8.7
Hz, H-2 and H-6) and 6.98 (d, J3,2 = J5,6 = 8.7 Hz, H-3 and H-5).
The 13C NMR spectrum of compound CBE3 in CDCl3 (Figure 138 and Table
27) showed signals for seven carbon atoms. The types of carbons are classified by the
analysis of the DEPT spectral spectrum as shown in Table 27.
All protons and carbons were assigned by analysis of the 1H-1H COSY,
HMQC and HMBC spectra (Figures 139-141). Compound CBE3 was identified as
the known compound 4-hydroxybenzaldehyde [226].
150
Table 27 NMR Spectral data of compound CBE3 (in CDCl3)
Compound CBE3
Position
*
1
H (mult., J in Hz)
13
C*
1
-
129.2 (C)
2
7.82 (d, 8.7)
132.5(CH)
3
6.98 (d, 8.7)
115.9 (CH)
4
-
161.6 (C)
5
6.98 (d, 8.7)
115.9 (CH)
6
7.82 (d, 8.7)
132.5 (CH)
4-OH
9.85 (s)
-
C=O
-
191.3
Carbon types were deduced from DEPT experiments.
151
1.21 Structure Determination of Compound CBE4
O
3
4
5
1'
2
1
O
3'
1''
2''
3'' OMe
2'
4''
6''
7
5''
OMe
6
OH
[227]
HRFABMS of compound CBE4 suggested a molecular formula of C18H20O5
from its [M+H]+ at m/z 317.1395 (calcd for 317.1389) corresponding to nine degrees
of unsaturation within the molecule. From the EIMS spectrum, the fragment ion at
m/z 194 could be formed by the rearrangement of the benzoate group and
phenylpropyl moiety (Figure 144 and Sheme 12). The IR spectrum demonstrated the
presence of a hydroxyl group at 3446 cm-1 (O-H stretching) and a carbonyl group at
1708 cm-1 (C=O stretching) (Figure 143). The UV spectral data exhibited absorption
bands at 228 and 272 nm (Figure 142).
The 1H NMR spectrum of compound CBE4 in CDCl3 (Figure 145 and Table
28) showed the presence of two benzene rings which was readily confirmed by
analysis of the 1H-1H COSY, HMQC and HMBC spectra (Figures 148-150). The 1H
NMR spectral data showed three methylene groups at δ 4.35 (H-1′), 2.09 (H-2′), 2.72
(H-3′) bridged between benzoate and aromatic rings, two methoxyls at δ 3.86 (each
3H, s) and one D2O-exchageable hydroxyl proton at δ 5.40 (br s) suggestive of the
phenylpropyl benzoate moiety with the substituents of hydroxyl and methoxyl on the
other aromatic ring (Figure 146).
In addition, HMBC data for compound CBE4 conclusively demonstrated
correlations of methylene protons at H-1′ to C-1 and H-2′ to C-1′′ and H-3′ to C-2′′
and C-1′ respectively. The 13C NMR spectrum of compound CBE4 in CDCl3 (Figure
147 and Table 28) showed the signal of carbonyl ester at δ 166.6. The position of two
methoxyl groups (3′′, 5′′-OMe) and one hydroxyl group (4′′-OH) were established
employing the HMBC technique as shown in Figure 7. All data are consistent with
the structure of compound CBE4 was thus assigned as a new compound, 3′-(4′′hydroxy-3′′,5′′-dimethoxyphenyl)-propyl benzoate [227].
152
Phenylpropyl benzoates have been previously reported as essential oil in
plants such as Wisteria floribunda (Ichiro et al., 1988). To our knowledge, the work
described here is the first report on phenylpropyl benzoates from plants in the genus
Croton.
Moreover, there has been no report on the biological activities of
phenylpropyl benzoate.
HMBC
1
H-1H COSY
O
3
5
2''
3'
2
4
1
O
1'
OMe
3''
1''
2'
6''
7
4''
OH
6
5''
OMe
Figure 7 1H-1H COSY and HMBC correlations of compound CBE4
+.
O
OMe
O
OH
OMe
m/z 316 (100%)
O+
+. H
O
OMe
O
OH
m/z 105 (30%)
OMe
-CO
+.
OH
+.
O
OMe
+
OH
OMe
m/z 77 (76%)
m/z 194 (84%)
-OCH3
OMe
+
OH
m/z 163 (75%)
Scheme 12 EIMS Spectra fragmentations of compound CBE4
153
Table 28 NMR Spectral data of compound CBE4 (in CDCl3)
Position
1
H (mult., J in Hz)
13
C*
Benzoate
1
-
166.6
2
-
130.3
3
8.05 (dd, 8.4, 1.3)
129.5
4
7.45 (tt, 7.4, 1.7)
128.3
5
7.57 (tt, 7.4, 1.3)
132.9
6
7.45 (tt, 7.4, 1.7)
128.3
7
8.05 (dd, 8.4, 1.3)
129.5
1′
4.35 (t, 6.5)
64.3
2′
2.09 (m)
30.5
3′
2.72 (br t)
32.5
1′′
-
132.3
2′′
6.43 (s)
104.9
3′′
-
146.9
4′′
-
132.9
5′′
-
146.9
6′′
6.43 (s)
104.9
3′′-OMe
3.86 (s)
56.2
4′′-OH
5.40 (s)
-
5′′-OMe
3.86 (s)
56.2
Propyl
Phenyl
*
Carbon types were deduced from DEPT experiments.
154
1.22 Structure Determination of Compound CBE5
O
3
4
5
1'
2
1
O
3'
1''
2''
2'
4''
6''
7
3'' OMe
5''
6
OH
[228]
Compound CBE5 was given the formula C17H18O4 from its [M+H]+ at m/z
287.1289 (calcd for 287.1284) in the HRFABMS. From the EIMS spectrum, the
fragment ion at m/z 164 could be formed by the rearrangement of the benzoate group
and phenylpropyl moiety (Figure 153 and Scheme 13). The IR spectrum exhibited
characteristic absorption bands at 3428 cm-1 (O-H stretching), 1718 cm-1 (C=O
stretching) (Figure 152). The UV spectrum revealed the absorption bands at 228 and
272 nm (Figure 151).
The 1H and
13
C NMR spectrum of compound CBE5 in CDCl3 (Figures 154-
155 and Table 29) were in good agreement with those of compound CBE4 except for
the absence of the signal of one of the methoxyl groups. The 1H NMR spectral data
of CBE5 showed peaks at δ 6.71 (1H, d, J = 1.3 Hz, H-2′′), δ 6.84 (1H, d, J = 8.3 Hz,
H-5′′) and δ 6.70 (1H, dd, J = 8.3, 1.3 Hz, H-6′′) ascribable to the three protons in an
ABX system of the 3′-phenyl- 3′′, 4′′-disubstituted ring system. On the basis of the
above spectroscopic data together with the 2D NMR such as the 1H-1H COSY,
HMQC and HMBC spectra (Figures156-158 and 8), compound CBE5 was identified
as 3′-(4′′-hydroxy-3′′-methoxyphenyl)-propyl benzoate or trivially known as
dihydroconiferyl benzoate [228]. This compound has already been isolated from the
flower of Gardenia taitensis DC (Lafontaine, Raharivelomanana and Bianchini,
1991), however no NMR spectral data have been reported.
155
HMBC
1
H-1H COSY
O
3
5
2''
3'
2
4
1
O
1'
3''
OMe
1''
2'
6''
7
4''
6
5''
OH
Figure 8 1H-1H COSY and HMBC correlations of compound CBE5
+.
O
O
OMe
OH
m/z 286 (100%)
O+
+. H
O
OMe
O
OH
m/z 105 (23%)
+.
OH
+.
O
OMe
+
OH
m/z 164 (100%)
m/z 77 (36%)
-OCH3
+
OH
m/z 133 (34%)
Scheme 13 EIMS Spectra fragmentations of compound CBE5
156
Table 29 NMR Spectral data of compound CBE5 (in CDCl3)
Position
1
H (mult., J in Hz)
13
C*
Benzoate
1
-
166.6
2
-
130.3
3
8.04 (dd, 8.4, 1.3)
129.5
4
7.44 (tt, 7.4, 1.7)
128.3
5
7.56 (tt, 7.4, 1.3)
132.9
6
7.44 (tt, 7.4, 1.7)
128.3
7
8.04 (dd, 8.4, 1.3)
129.5
1′
4.34 (t, 6.5)
64.2
2′
2.07 (m)
30.5
3′
2.72 (br t)
31.9
1′′
-
133.0
2′′
6.71 (d, 1.3)
120.9
3′′
-
146.4
4′′
-
143.8
5′′
6.84 (d, 8.3)
114.3
6′′
6.70 (dd, 8.3, 1.3)
110.9
3′′-OMe
3.84 (s)
55.8
4′′-OH
5.59 (br s)
-
Propyl
Phenyl
*
Carbon types were deduced from DEPT experiments.
157
1.23 Structure Determination of Compound CBE6
O
3
1'
2
4
5
1
O
2'
3'
1''
2''
3''
4''
6''
7
OH
5''
6
[229]
Compound CBE6 had a molecular formula of C16H16O3 from its [M+H]+ at
m/z 257.1178 (calcd for 257.1179) based on HRFABMS. From the EIMS spectrum,
the fragment ion at m/z 134 was formed by the rearrangement of the benzoate group
and phenylpropyl moiety (Figure 161 and Scheme 14). The UV absorptions exhibited
the absorption bands at 228 and 279 nm (Figure 159). The IR spectrum revealed the
absorption bands at 3377 cm-1 (O-H stretching) and carbonyl group at 1698 cm-1
(C=O stretching) (Figure 160).
The 1H and 13C NMR data of compound CBE6 in CDCl3 (Figure 162-163 and
Table 30) was similar to those of compound CBE6 except for the absence of two
methoxyl groups at C-5′′ and C-3′′. The 1H NMR spectral data of compound CBE6
showed the presence of the para-substituted benzene ring at δ 7.08 (2H, d, J = 8.4 Hz,
H-2′′ and H-6′′) and δ 6.77 (2H, d, J = 8.4 Hz, H-3′′ and H-5′′). The 1H-1H COSY,
HMQC and HMBC experiment on CBE6 (Figures 164-166 and 9) also produced very
similar results, indicating that compound CBE4, CBE5 and CBE6 were closely
related. Based on the above spectral evidence, compound CBE6 was identified as a
new compound and has been named 3′-(4′′-hydroxyphenyl)-propyl benzoate [229].
HMBC
1
H-1H COSY
O
3
5
7
6
2''
3'
2
4
1
O
1'
2'
3''
1''
6''
4''
5''
OH
Figure 9 1H-1H COSY and HMBC correlations of compound CBE6
158
O
+.
O
OH
m/z 258 (3%)
O+
+. H
O
O
OH
m/z 105 (50%)
+.
OH
+.
O
+
OH
m/z 134 (38%)
m/z 77 (36%)
-H
+
OH
m/z 133 (100%)
Scheme 14 EIMS Spectra fragmentations of compound CBE6
159
Table 30 NMR Spectral data of compound CBE6 (in CDCl3)
Position
1
H (mult., J in Hz)
13
C*
Benzoate
1
-
166.8
2
-
130.3
3
8.04 (dd, 8.6, 1.1)
129.5
4
7.45 (tt, 7.5, 1.6)
128.4
5
7.57 (tt, 7.5, 1.1)
132.9
6
7.45 (tt, 7.5, 1.6)
128.4
7
8.04 (dd, 8.6, 1.1)
129.5
1′
4.33 (t, 6.5)
64.3
2′
2.07 (m)
30.5
3′
2.72 (br t)
31.3
1′′
-
133.2
2′′
7.08 (d, 8.4)
129.5
3′′
6.77 (d, 8.4)
115.3
4′′
-
153.9
5′′
6.77 (d, 8.4)
115.3
6′′
7.08 (d, 8.4)
129.5
4′′-OH
5.59 (br s)
-
Propyl
Phenyl
*
Carbon types were deduced from DEPT experiments.
160
2. Biological Activities of Compounds from Bauhinia sirindhorniae
2.1 Antimicrobial Activity
The crude extracts obtained from Bauhinia sirindhorniae were examined for
this activity against Staphylococcus aureus ATCC 29213, Bacillus subtilis ATCC
6633, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922,
Candida albicans ATCC 10231 and Trichophyton mentagrophytes (clinical isolated).
It was found that some crude extracts possessed antibacterial activities. The 95%
ethanol extracts of stems and roots showed activities against Bacillus subtilis ATCC
6633 and Staphylococcus aureus ATCC 29213. The 95% ethanol extracts of the
stems showed inhibition zone diameters of 18.55 and 15.55 mm, respectively and the
same extracts of the roots showed inhibition zone diameters of 16.05 and 13.60 mm,
respectively. Isoliquiritigenin [14], (+)-isolariciresinol-3α-O-α-L-rhamnoside [215],
trimethoxyphenolic-1-O-β-D-glucoside [216], lithospermoside [54], (2S)-naringenin
[17], luteolin [220], (2S)-eriodictyol [16], (+)-lyoniresinol-3α-O-α-L-rhamnoside
[222] and menisdaurin [224] from the extracts of the roots and stems were subjected
to determination of the MIC and MBC. Studies of antibacterial activity of the isolated
compounds are shown in the Table 31. It was found that (2S)-eriodictyol [16] and
isoliquiritigenin [14] showed the activity against B. subtilis. Anti-S. aureus activity of
both compounds was found to be equal in term of MIC at 200 µg/ml.
Naringenin [17] and luteolin [220] exhibited activity against B. subtilis.
(2S)-
161
Table 31 Antibacterial activity of some isolated compounds from Bauhinia
sirindhorniae
Compound
S. aureus ATCC 29213
B. subtilis ATCC 6633
MIC
MBC
MIC
MBC
[14]
200
>200
100
100
[215]
NA
NA
NA
NA
[216]
NA
NA
NA
NA
[54]
NA
NA
NA
NA
[17]
NA
NA
100
>200
[220]
NA
NA
200
200
[16]
200
200
50
>200
[222]
NA
NA
NA
NA
[224]
NA
NA
NA
NA
Penicillin G
0.0625
0.0625
0.031
0.031
MIC: Minimum Inhibitory Concentration (µg/ml)
MBC: Minimum Bactericidal Concentration (µg/ml)
NA: No Activity
2.2 Free Radical Scavenging Activity
By TLC screening assay, the 95% ethanol extracts from stems and roots of
Bauhinia sirindhorniae showed free radical scavenging activity. The free radical
scavenging activity was evaluated as IC50 and Trolox equivalent antioxidant capacity
(TEAC) for some isolated compounds from B. sirindhorniae that have not been
reported before. (+)-Isolariciresinol-3α-O-α-L-rhamnoside [215], (+)-lyoniresinol3α-O-α-L-rhamnoside [222], lithospermoside [54] and menisdaurin [224] were
subjected for this activity.
The cyanoglucosides, lithospermoside [54] and
menisdaurin [224] showed very weak free radical scavenging activity and thus the
TEAC value and IC50 values could not be determined. The lignan glycosides, (+)isolariciresinol-3α-O-α-L-rhamnoside
[215]
and
(+)-lyoniresinol-3α-O-α-L-
rhamnoside [222] showed moderate activity in comparison with quercetin as a
positive control, as shown in Table 32.
162
OH
OH
HO
O
OH
OH
.
O
Quercetin
Table 32 The DPPH radical scavenging activity of compounds [222] and [215]
Compound
TEAC*
IC50 (µM)
[222]
0.95
67
[215]
0.99
76
Quercetin
1.91
17
*TEAC: Trolox Equivalent Antioxidant Capacity
3. Biological Activities of Compounds from Croton hutchinsonianus
3.1 Cytotoxic Activity
Preliminary bioactivity screening revealed that Croton hutchinsonianus
exhibited cytotoxic activity. The results are summarized in Table 33.
Table 33 The cytotoxic activity against NCI H-187 cell lines of the crude extracts
of Croton hutchinsonianus
Activity
IC50 (µg/ml)
The hexane leaves extract
Moderately active
5.8
The ethyl acetate leaves extract
Moderately active
8.6
The 95% ethanol leaves extract
Inactive
-
The hexane branches extract
Strongly active
1.1
The ethyl acetate branches extract
Weakly active
13.8
The 95% ethanol branches extract
Inactive
-
Crude extract
The compounds investigated for cytotoxic activity were 3′-(4′′-hydroxy-3′′,5′′dimethoxyphenyl)-propyl benzoate [227], dihydroconiferyl benzoate [228] and 3′-
163
(4′′-hydroxyphenyl)-propyl benzoate [229] , all of which were isolated from C.
hutchinsonianus.
3′-(4′′-Hydroxy-3′′,5′′-dimethoxyphenyl)-propyl benzoate [227] displayed
weak cytotoxic activity with the IC50 of 11.38 µg/ml while dihydroconiferyl benzoate
[228] and 3′-(4′′-hydroxyphenyl)-propyl benzoate [229] were inactive.
3.2 Antifungal Activity
3′-(4′′-Hydroxy-3′′,5′′-dimethoxyphenyl)-propyl
benzoate
[227],
dihydro
coniferylbenzoate [228] and 3′-(4′′-hydroxyphenyl)-propyl benzoate [229] isolated
from C. hutchinsonianus, were subjected to biological evaluation for antifungal
activity against Candida albicans.
3′-(4′′-Hydroxy-3′′,5′′-dimethoxyphenyl)-propyl benzoate [227], dihydro
coniferyl benzoate [228] and 3′-(4′′-hydroxyphenyl)-propyl benzoate [229] showed
moderate antifungal activity with the IC50 of 12.43, 7.48 and 5.35 µg/ml, respectively.
It should be noted that phenylpropyl benzoate displayed antifungal activity against
C. albicans.
CHAPTER V
CONCLUSION
The present investigation deals with the isolation of several biogenetically
related compounds from the stems and roots of Bauhinia sirindhorniae K. & S.S.
Larsen. Two cyanoglucosides (lithospermoside [54] and menisdaurin [224]), one
flavan ((-)-epicatechin [217]), two flavanones ((2S)-naringenin [17] and (2S)eriodictyol [16]), one flavanonol ((+)-taxifolin [221]), one flavone (luteolin [220]),
one chalcone (isoliquiritigenin [14]), one chromone (5,7-dihydroxychromone [219]),
one chromone glucoside (5-hydroxychromone 7-β-D-glucoside [223]), two lignan
glycosides ((+)-isolariciresinol-3α-O-α-L-rhamnoside [215] and (+)-lyoniresinol-3αO-α-L-rhamnoside [222]), two triterpenoids (lupeol [77] and glutinol [214]), one
steroid glucoside (sitosteryl-3-O-β-D-glucoside [37]) and other phenolic compounds
(3,4,5-trimethoxyphenolic-1-O-β-D-glucoside [216] and protocatechuic acid [218])
were isolated.
Scavenging activity of some isolated compounds from B.
sirindhorniae towards DPPH (1,1-diphenyl-2-picrylhydrazyl) radical was also
described. The lignan glycosides ((+)-isolariciresinol-3α-O-α-L-rhamnoside [215]
and (+)-lyoniresinol-3α-O-α-L-rhamnoside [222]) showed moderate activity in
comparison with quercetin as a positive control.
(2S)-Eriodictyol [16] and
isoliquiritigenin [14] showed activity against Bacillus subtilis and Staphylococcus
aureus whereas (2S)-naringenin [17] and luteolin [220] exhibited activity against
Bacillus subtilis.
Chemical examination of the branches and leaves of Croton
hutchinsonianus Hosseus led to the isolation of two new compounds 3′-(4′′-hydroxy3′′,5′′-dimethoxyphenyl)-propyl benzoate [227] and 3′-(4′′-hydroxyphenyl)-propyl
benzoate [229] and other four known compounds, namely farnesyl acetone [225],
poilaneic acid [163], 4-hydroxybenzaldehyde [226] and dihydroconiferylbenzoate
[228].
The isolated compounds from C. hutchinsonianus were subjected for
biological activities evaluation, involving antifungal activity and cytotoxicity. 3′-(4′′Hydroxy-3′′,5′′-dimethoxyphenyl)-propyl benzoate [227], dihydroconiferyl benzoate
[228] and 3′-(4′′-hydroxyphenyl)-propyl benzoate [229] revealed moderate antifungal
activity
against
Candida
albicans.
In
addition,
3′-(4′′-hydroxy-3′′,5′′-
dimethoxyphenyl)-propyl benzoate [227] showed weak cytotoxic activity against
165
NCI-H187 cell line while dihydroconiferylbenzoate [228] and 3′-(4′′-hydroxyphenyl)propyl benzoate [229] were inactive.
Table 34 Compounds isolated from chloroform extract of the stems of Bauhinia
sirindhorniae
Compound
Antibacterial Activity
Free Radical Scavenging
Activity
Triterpenes
Lupeol [77]
ND
ND
Glutinol [214]
ND
ND
ND: Not Determined
Table 35 Compounds isolated from butanol extract of the stems of Bauhinia
sirindhorniae
Compound
Antibacterial Activity
Free Radical Scavenging
Activity
Chalcone
Isoliquiritigenin [14]
Active
ND
Inactive
Active
ND
ND
ND
Inactive
ND
ND
Inactive
Inactive
Lignan Glycoside
(+)-Isolariciresinol-3α-O-αL-rhamnoside [215]
Flavan
((-)-Epicatechin [217]
Phenolic Compounds
3,4,5-Trimethoxyphenolic-1O-β-D-glucoside [216]
Protocatechuic acid [218]
Cyanoglucoside
Lithospermoside [54]
166
Table 36 Compounds isolated from chloroform extract of the roots of
Bauhinia sirindhorniae
Compound
Antibacterial Activity
Free Radical Scavenging
Activity
Chromone
5,7-Dihydroxychromone [219]
ND
ND
ND
ND
Steroid Glycoside
Sitosteryl-3-O-β-D-glucoside [37]
Table 37 Compounds isolated from butanol extract of the roots of Bauhinia
sirindhorniae
Compound
Antibacterial Activity
Free Radical Scavenging
Activity
Flavone
Active
ND
(2S)-Naringenin [17]
Active
ND
(2S)-Eriodictyol [16]
Active
ND
ND
ND
Inactive
Active
ND
ND
Inactive
Inactive
Luteolin [220]
Flavanones
Flavanonol
(+)-Taxifolin [221]
Lignan Glycoside
(+)-Lyoniresinol-3α-O-α-Lrhamnoside [222]
Chromone Glucoside
5-Hydroxychromone 7-β-Dglucoside [223]
Cyanoglucoside
Menisdaurin [224]
167
Table 38 Compounds isolated from
ethyl acetate extract of Croton
hutchinsonianus
Cytotoxic Against
Antifungal Against
NCI H-187
Candida albicans
ND
ND
ND
ND
ND
ND
Weakly Active
Moderately Active
Dihydroconiferyl benzoate [228]
Inactive
Moderately Active
3′-(4′′-Hydroxyphenyl)-propyl
Inactive
Moderately Active
Compound
C18 Terpenoid
Farnesyl acetone [225]
Diterpene
Poilaneic acid [163]
Benzaldehyde
4-Hydroxybenzaldehyde [226]
Phenylpropyl Benzoates
3′-(4′′-Hydroxy-3′′,5′′dimethoxyphenyl)-propyl
benzoate [227]
benzoate [229]
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