View metadata, citation and similar papers at core.ac.uk
brought to you by
CORE
provided by KhartoumSpace
Phytochemical and Antifungal Analysis of Extracts of
Terminalia brownie (Fresen. Mus. Senckenb) Shaf
By
Enass Yousif Abdelkarim Salih
B.Sc. (Forestry. Honor)
University of Khartoum
(2003)
A dissertation Submitted in Fulfillment of the Requirements for
the Degree of Master of Science in Forestry to the University of
Khartoum
Supervisor
Dr. Hiba Abdel Rahman Ali
Department of Forest Products and Industries
Faculty of Forestry
University of Khartoum
July 2009
DEDICATION
To the sunshine in my life Mum
To Soul of my Father
To my Sisters and brothers
To my Colleagues
I dedicated this work
i
ACKNOWLEDGEMENTS
Thanks to Ulha who gave me strength, patience, health and insistence to
complete my study.
I would like to express my sincere gratitude to my supervisor, Dr. Hiba
Abdel Rahman Ali for the encouragement, exceptional ideas, and tireless
optimism that have kept me going. I am also grateful for my cosupervisor Dr. Ashraf Mohammed Ahmed Ubdulla for his good efforts
and support during my work. I owe thanks to my Godfather Dr
Abdelazim Yassin dean of the faculty of forestry university of Khartoum.
My warm thanks are due to Bashier Abaker, Hassan Gumaa Rajah and.
Ubdallha Saaid Salim for their botanical expertise, and Hythem Hashim
for useful scientific hints as a plant taxonomist.
I am especially grateful to Dr Nagla and Aymen Ashmyg for their fruitful
co-operation during the research of the biological activity tests. Mai
Hassan deserves genial thanks for support.
I want to express my sincere thanks to all my colleagues at the
Department of Forest Products and Industries and to the personnel at the
Commission of Biotechnology and Genetic Engineering, National Center
of the Research for their co-operation.
Our deepest thank goes to Prof Nickoli Kuhnert and his group, School of
Engineering and Science, International University, Bremen, Germany for
assisting us in running the Tandem Mass Spectra.
My most profound appreciation goes to my lovely family, Mohammed,
Omima ,Nasir and Amel. Special thanks goes to the affectionate woman
(mum) for her great impact in my life.
ii
TABLE OF CONTENTS
DEDICATION………………………………………………………..
i
ACKNOWLEDGMENTS…………………………………………….
ii
TABLE OF CONTENTS……………………………………………...
iii
ABBREVIATIONS…………………………………………………...
v
LIST OF TABLES……………………………………………………...
vi
LIST OF FIGURES…………………………………………………...
vii
ABSTRACT…………………………………………………………...
viii
ARABIC ABSTRACT……………………………………………….
x
CHAPTER ONE
1
1. Introduction…………………………………………………………
1
1.1 Objectives of this study……………………………………………..
3
CHAPTER TWO
4
2. Literature Review…………………………………………………
4
2.1 Taxonomy of genus Terminalia ……………………………………
4
2.2 Antimicrobial metabolites of plant origin ...…………………………
5
2.3 Combretaceae secondary metabolites and their biological significance...
6
2.4 Terminalia important secondary metabolites...
6
2.5 Plant pathogens tested.....
18
2.6 Biosynthesis of Flavonoids........................................
20
2.7 Chromatographic analysis of flavonoids ……………………
23
2.8 Stilbenes and their biosynthetic pathway........
24
2.9 Chromatographic analysis of stilbenes and phenanthrenes …………
28
CHAPTER THREE
29
Material and Methods ………………………………………………
29
3.1 Plant material collection………………………………………….
29
3.2 Plant material preparation and extraction…………………………
31
3.3. Chromatography………………………………………………….
33
iii
3.3.1 Thin layer chromatography……………………………………...
33
3.3.1.1 Spray reagents…………………………………………………
35
3.4.2 Solid phase extraction. …………………………………………
35
3.4.3 High performance liquid chromatography (HPLC)……………..
36
3.4.4 Triple quadruple mass spectrometric analysis (MS/MS)………
36
3.5 Antimicrobial activity……………………………………………..
37
3.5.1 Preparation of fungal suspensions………………………………
37
3.5.2 Testing for antifungal activity cup- plate Agar diffusion method.........
37
3.5.3 Minimum inhibition concentration (MIC) ………………
38
CHAPTER FOUR
40
Results and Discussion…………………………………………..........
40
4.1 Thin layer chromatography of the bark and the wood of T. brownii…..
40
4.2 Antimicrobial activity of T. brownii ……………………………..
46
4.3 Minimum inhibition concentration………………………………...
49
4.4 RP-HPLC-DAD of T. brownii wood ethyl acetate phase …………….……
50
4.4.1 Identification of compounds in T. brownii bark and wood ethyl acetate
54
phase by LC-triple quadruple mass spectrometric analysis(LC-MS/MS)………
4.4.2 Compounds structures assignment in the ethyl acetate phase of the wood
of T. brownii
56
………………………………………………………………..
CHAPTER FIVE
61
Conclusion and Recommendations...
61
5.1 Conclusion...
61
5.2 Recommendations...
62
CHAPTER SIX
64
References……………………………………………………......
Appendix……………………………………………………………
iv
64
ABBREVIATIONS
CHCL3
CID
DAD
DPPH
ESI
EtoAc
HCl
HCO2H
HPLC
H2SO4
Marc
MeCN
MeOH
MIC
MS
MS/MS
m/z
µl
mm
NP
NPR
PE
PEG
Rf
RP
Rt
S.D.A
SPE
SR
TLC
Chloroform
Collision induced dissociation
Diode array detector
1, 1-Diphenyl-2-pierylhydrazyle
Electrospray Ionization
Ethyl acetate
Hydrochloric acid
Formic acid
High Performance Liquid Chromatography
Sulphuric acid
Residue of the extract
Acetonitrile
Methanol
Minimum inhibition concentration
Mass spectroscopy
Tandem mass Spectroscopy
Mass to charge ratio
Micromilliter
Millimeter
Normal phase
Natural products reagent
Petroleum ether
Polyethylenglycol
Retardation factor
Reverse phase
Retention time
Sabouroud dextrose agar
Solid Phase extraction
Spray reagents
Thin layer chromatography
Ultra Violet
UV
ג
Wavelength (nm)
v
List of Tables
No.
Name
Page
1
African and Sudanese traditional uses of Terminalia spp
17
2
Developing solvent system used in TLC
34
TLC profile of the ethyl acetate fraction of T. brownii bark
3
and wood sprayed with vanillin H2SO4 reagent
42
TLC profile of the ethyl acetate fraction of T. brownii Bark
4
and wood sprayed with NPR reagent
43
Antimicrobial activity of the wood and bark extracts of T.
5
48
brownii
Peak No. (Fig 11), RP- HPLC data (Rt), molecular weight
6
(m/z), MS/MS data (m/z) and assigned structures of the wood
of T. brownii ethyl acetate fraction
vi
60
List of Figures
No.
Name
Page
A- Stilbenes reported in Combretaceae
10
B- Flavonoids reported in Terminalia spp.
11
C- Terpenes reported in Terminalia spp.
14
2
Biosynthesis of the different classes of flavonoids
22
3
Stilbenes biosynthesis
27
4
Terminalia brownii parts
30
5
Schematic diagram of T. brownii wood and bark extraction
32
1
A- TLC Profile in normal phase silica gel of the ethyl acetate
6
phases of the studied parts of T. brownii. (A) 254nm, (B)
44
Sprayed with NPR at 366nm
B- TLC Profile in reversed phase of the ethyl acetate phases of
the studied parts of T. brownii. Sprayed with NPR (A) 254 and
45
(B) 366 nm
RP-HPLC-DAD Chromatogram of the ethyl acetate fraction of
7
T. brownii wood (A) and bark (B) recorded at λmax 254nm
51
RP-HPLC-DAD Chromatogram of the ethyl acetate fraction of
8
T. brownii bark (A) and wood (B) recorded at λmax 320-380 nm
52
RP- HPLC-DAD chromatogram of the ethyl acetate fraction of
9
T. brownii wood recorded at λmax 254(A) and 320(B) nm
53
Fragmentation pathways for flavonoid glycosides (illustrated on
10
55
apigenin-7-O-rutinoside)
RP-HPLC-DAD Chromatogram of the ethyl acetate fraction of
11
T.brownii wood extract at λmax 320-380 nm
vii
59
Title: Phytochemical and Antifungal Analysis of Extracts
of Terminalia brownii (Fresen. Mus. Senckenb)(ٍٍShaf)
Name: Enass Yousif Abdelkarim
Abstract:
Combretaceae is known for its medical uses in Africa and Asia. This
study was conducted for phytochemical analysis of two different parts of
Terminalia brownii (Combretaceae). Plant studied wood and bark
extracts were subjected to biological and chemical screening
implementing different chromatographic analytical methods (TLC, HPLC
and LC-MS/MS)
Antimicrobial activity of the different extracts of T. brownii (wood, bark)
was recorded against different plant pathogenic fungi. The aqueous
extract of the wood of T. brownii exhibited the highest antifungal activity
against Aspergillus niger (13mm), 11mm inhibitory zone against A.
flavus and Natrassia mangifera
and 12mm inhibition zone against
Fusarium moniliform. The inhibitory zones of the aqueous extract of the
bark were 14mm against A. niger and A. flavus, and 20mm against N.
mangifera and F. moniliform. The ethyl acetate extract of the wood and
bark of T. brownii gave similar growth inhibitory zones with the mean
diameter of 15mm against A. niger, A. flavus, N. mangifera and F.
moniliform.
Minimum inhibition concentration of the ethyl acetate extracts of the
wood and the bark of T. brownii was measured against the four tested
plants pathogens. MIC could not be determined because even at
concentration as low as, 0.001g/ml antifungal activity was observed. MIC
could be considered to be lower than 0.001g/ml. Oppositely; no effect
was shown for the same extracts against A. niger even at the higher
viii
concentration (0.05g/ml). This result demonstrated that the ethyl acetate
extracts of the wood and the bark of T. brownii against A. niger are either
not effective or they may have an MIC above 0.05g/m.
Thin layer chromatography (TLC) revealed absences of alkaloids in the
extracts of the different parts. Flavonoids and stilbenes were mainly
accumulated in the ethyl acetate fraction of both studied parts (wood and
bark). Terpenoids were detected in all extracts screened.
Reverse phase HPLC coupled with UV detector (RP-HPLC-DAD)
proved the presence of flavonoids and flavonoidal acids in the different
parts studied. The flavonoids detected were mainly flavonones, flavonols
and their derivatives. Similar compounds exist among the active extracts,
namely the ethyl acetate phases of the wood and bark of the plant studied.
Accordingly, the wood ethyl acetate fraction was subjected to further
analysis for identification of the major compounds with special emphasis
on flavonoids and stilbenes content.
Tandem mass spectrometry (MS/MS) led to the identification of ten
compounds three of which were pentacyclic triterpenoidal acids namely
masilinic acid, asistic acid and arjunic glycoside. Resveratrol 3-O-βgalloylglucoside was also identified in its cis and trans forms. Flavonoids
identified in T. browni wood ethyl acetate extract include two Quercitin
derivatives namely Quercitin 7-O-β- diglucoside and Quercitin 7-Ogalloylglucoside. Naringenin 4’methoxy 7 arabinoside together with
Naringenin 7 ellagic acid were detected in this extract. Additionally, 5,6
dihydroxy 3’4’7 trimethoxy flavone was among the identified flavonoids
in this extract.
These results support the final aim of using the extracts of T. brownii
wood and bark to biologically control plants pathogenic fungi.
ix
Title: Phytochemical and Antifungal Analysis of Extracts of
)Terminalia brownii (Fresen. Mus. Senckenb)(Shaf
Name: Enass Yousif Abdelkarim
ﺧﻼﺻﺔ اﻻﻃﺮوﺣﺔ
ﻟﻬﺎ اﺳﺘﻌﻤﺎﻻت ﻃﺒﻴﺔ ﻓﻲ ﻗﺎرﺗﻲ اﻓﺮﻳﻘﻴﺎ واﺳﻴﺎ .هﺬﻩ اﻟﺪراﺳﺔ ﺧﻠﺼﺖ Combertaceaceﻋﺎﺋﻠﺔ
اﻟﻲ اﻟﺘﺤﻠﻴﻞ اﻟﻜﻴﻤﻴﺎﺋﻲ ﻟﺠﺰﺋﻴﻦ ﻣﻦ اﻟﺸﺎف .
دراﺳﺔ ﻣﺴﺘﺨﻠﺺ اﻟﻠﺤﺎء واﻟﺨﺸﺐ ﻓُﻌﻠﺖ ﺑﻴﻮﻟﻮﺟﻴﺎ وآﻴﻤﻴﺎﺋﻴًﺎ ﺑﺘﻄﺒﻴﻖ ﻣﺨﺘﻠﻒ ﻃﺮق اﻟﺘﺤﻠﻴﻞ
اﻟﻜﺮوﻣﻮﺗﻮﻏﺮاﻓﻲ )آﺮوﻣﻮﺗﻮﻏﺮاﻓﻴﺎ اﻟﻄﺒﻘﺔ اﻟﺮﻗﻴﻘﺔ ،وآﺮوﻣﻮﺗﻮﻏﺮاﻓﻴﺎ اﻟﻀﻐﻂ اﻟﻌﺎﻟﻲ وﻣﻜﺸﺎف
اﻟﻄﻴﻒ اﻟﻀﻮﺋﻲ(.
اﻟﻤﻜﺎﻓﺤﺔ اﻟﺤﻴﻮﻳﺔ ﻟﻠﻤﺴﺘﺨﻠﺼﻴﻦ )اﻟﺨﺸﺐ واﻟﻠﺤﺎء( .ﻓُﻌﻠﺖ ﺿَﺪ آﺎﺋﻨﺎت ﻧﺒﺎﺗﻴﺔ ﻣﻤﺮﺿﺔ ﻣﻦ اﻧﻮاع
ﻞ أﻋﻠﻲ ﻣﻨﻄﻘﺔ ﻟﺘﺜﺒﻴﻂ اﻟﻨﻤﻮ اﻟﻔﻄﺮي ﻓﻲ ﻓﻄﺮ
ﻣﺨﺘﻠﻔﺔ .اﻟﻤﺴﺘﺨﻠﺺ اﻟﻤﺎﺋﻲ ﻟﺨﺸﺐ اﻟﺸﺎف ﺳﺠ َ
و ﻓﻄﺮﻳﺎت اﻟﻤﻮت اﻟﺘﺮاﺟﻌﻲ 13 )A.favusﻣﻠﻢ( ،و11ﻣﻠﻢ ﻣﻨﻄﻘﺔ ﺗﺜﺒﻴﻂ ﺿَﺪ A.niger
Fusarium moniliform .و 12ﻣﻠﻢ ﻣﻨﻄﻘﺔ ﺗﺜﺒﻴﻂ ﺿَﺪ Nattrassia mangifera
و A.flavus 20و A.nigerﻣﻨﻄﻘﺔ ﻣﻨﻊ اﻟﻨﻤﻮاﻟﻔﻄﺮي ﻓﻲ اﻟﻤﺴﺘﺨﻠﺺ اﻟﻤﺎﺋﻲ ﻟﻠﺤﺎء 14ﻣﻠﻢ ﺿًﺪ
F.moniliform .و N.mangiferaﻣﻠﻢ ﻣﻨﻄﻘﺔ ﺗﺜﺒﻴﻂ اﻟﻨﻤﻮ ﻓﻲ ﻓﻄﺮ
ﻣﺴﺘﺨﻠﺺ ﺧﻼت اﻻﻳﺜﻴﻞ ﻟﺨﺸﺐ وﻟﺤﺎء اﻟﺸﺎف أﻋﻄﻲ ﻧﺘﺎﺋﺞ ﻣﺘﺸﺎﺑﻪ ﻓﻲ ﻣﻨﻄﻘﺔ ﺗﺜﺒﻴﻂ اﻟﻨﻤﻮ اﻟﻔﻄﺮي
ﻣﻊ ﻣﺘﻮﺳﻂ ﻗﻄﺮ ﻣﻘﺪارﻩ 10ﻣﻠﻢ ﺿَﺪ آﻞ ﻣﻦ F.moniliform, A.flavus , A.niger
. N.mangifera,أﻗﻞ ﺗﺮآﻴﺰ ﻓﻲ ﻣﺴﺘﺨﻠﺺ ﺧﻼت اﻻﺛﻴﻞ ﻟﺨﺸﺐ وﻟﺤﺎء اﻟﺸﺎف ﻗﻴﺴﺖ ﺿَﺪ أرﺑﻊ
آﺎﺋﻨﺎت ﻣﻤﺮﺿﺔ ﻧﺒﺎﺗﻴ ًﺎ .أﻗﻞ ﺗﺮآﻴﺰ ﻣﻨﻊ اﻟﻨﻤﻮ اﻟﻔﻄﺮي ﻟﻢ ﻳﺤﺪَد ﺑﻌﺪ ﻻﻧﻪ ﻓﻲ أﻗﻞ ﺗﺮآﻴﺰ ) 0.001ﺟﻢ
ﻣﻠﻢ( ﻟﻮﺣﻆ ﻋﺪم اﻟﻨﻤﻮ اﻟﻔﻄﺮي .أﻗﻞ ﺗﺮآﻴﺰ ﻳﻤﻨﻊ اﻟﻨﻤﻮ اﻟﻔﻄﺮي ﻳﺠﺐ ان ﻳﻜﻮن اﻗﻞ ﻣﻦ 0.001ﺟﻢ
/ﻣﻠﻢ .إﻳﺠﺎﺑﻴًﺎ ﻳﻼﺣﻆ ﻧﻔﺲ اﻟﺘﺄﺛﻴﺮ ﻟﻠﻤﺴﺘﺨﻠﺺ ﺿَﺪ A.nigerﺣﺘﻲ ﻓﻲ أﻋﻠﻲ ﺗﺮآﻴﺰ
ﻟﻠﻤﺴﺘﺨﻠﺺ0.05ﺟﻢ /ﻣﻞ .هﺬﻩ اﻟﻨﺘﻴﺠﺔ أﻇﻬﺮت أن ﻣﺴﺘﺨﻠﺺ اﻟﺨﻼت اﻻﻳﺘﻴﻠﻲ ﻟﺨﺸﺐ وﻟﺤﺎء
اﻟﺸﺎف ﺿَﺪ A.nigerإﻣﺎ ﻟﻴﺲ ﻟﻪ ﺗﺎﺛﻴﺮ أو ﻳﺤﺪث ﺗﺎﺛﻴﺮ ﻋﻨﺪﻣﺎ ﻳﻜﻮن أﻗﻞ ﺗﺮآﻴﺰ ﻳﺜﺒﻂ اﻟﻨﻤﻮ
اﻟﻔﻄﺮي أﻋﻠﻲ ﻣﻦ 0.05ﺟﻢ /ﻣﻞ .
ﻋﻨﺪ ﺗﺘﻄﺒﻴﻖ آﺮوﻣﺎﺗﻮﻏﺮاﻓﻴﺎ اﻟﻄﺒﻘﺔ اﻟﺮﻗﻴﻘﺔ ﻟﻮﺣﻆ ﻏﻴﺎب اﻟﻘﻠﻮﻳﺎت ﻓﻲ ﻣﺴﺘﺨﻠﺺ اﻟﺨﺸﺐ واﻟﻠﺤﺎء.
ﻟﻮﺣﻆ وﺟﻮد اﻟﻔﻼﻓﻮﻧﻮﻳﺪ و اﻻﺳﺘﻠﺒﻴﻦ ﺑﺼﻮرة اﺳﺎﺳﻴﺔ ﻓﻲ ﻣﺴﺘﺨﻠﺺ اﻟﺨﻼت اﻻﺛﻴﻠﻲ ﺑﺎﻟﺨﺸﺐ
واﻟﻠﺤﺎء .آﻞ اﻟﻤﺴﺘﺨﻠﺼﺎت أﻇﻬﺮت وﺟﻮد اﻟﺘﺒﺮﺑﻨﻮﻳﺪات .
x
آﺮوﻣﺎﺗﻮﻏﺮاﻓﻴﺎ اﻟﻀﻐﻂ اﻟﻌﺎﻟﻲ ﻟﻠﻄﻮر اﻟﻌﻜﺴﻲ ﺟُﻤﻌﺖ ﻣﻊ اﻟﻤﻜﺸﺎف اﻟﻄﻴﻔﻲ وﺑﺮهﻨﺖ وﺟﻮد
اﻟﻔﻼﻓﻮﻧﻴﺪ واﺣﻤﺎض اﻟﻔﻼﻓﻮﻧﻴﺪل ﻓﻲ ﻣﺨﺘﻠﻒ اﺟﺰاء اﻟﺪراﺳﺔ .واﻟﻔﻼﻓﻮﻧﻴﺪات اﻟﺘﻲ ﻇﻬﺮت هﻲ
اﻟﻤﺘﺸﺎﺑﺔ ﺗﻮﺟﺪ ﻓﻲ اﻟﻤﺴﺘﺨﻠﺼﺎت اﻟﻨﺸﻄﺔ ﺗﺤﺪﻳﺪًا اﻟﻔﻼﻓﻮﻧﻮن واﻟﻔﻼﻓﻮﻧﻮل وﻣﺸﺘﻘﺎﺗﻬﻢ .اﻟﻤﺮآﺒﺎت
ﻣﺴﺘﺨﻠﺺ اﻟﺨﻼت اﻻﺛﻴﻠﻲ ﻟﺨﺸﺐ وﻟﺤﺎء اﻟﺸﺎف .ﻃﺒﻘَﺎ ﻟﺬﻟﻚ ﺟُﺰء ﻣﺴﺘﺨﻠﺺ ﺧﻼت اﻻﺛﻴﻞ اﻟﺨﺸﺒﻲ
وﻓﻌًﻞ ﻟﺘﺤﺎﻟﻴﻞ اﺑﻌﺪ ﻟﻠﺘﻌﺮف ﻋﻠﻲ اﻟﻤﺮآﺒﺎت اﻟﺮﺋﻴﺴﻴﺔ ﻣﻊ اﻟﺘﺮآﻴﺰ ﻋﻠﻲ ﻣﺤﺘﻮاهﺎ ﻣﻦ اﻟﻔﻼﻓﻮﻧﻴﺪ
واﻻﺳﺘﻠﺒﻴﻦ .
ﺗﺠﺎرب ﻣﻄﺎﻓﻴﺔ اﻟﻀﻮء ﺁدت اﻟﻲ اﻟﺘﻌﺮف ﻋﻠﻲ ﻋﺸﺮة ﻣﺮآﺒﺎت آﻴﻤﻴﺎﺋﻴﺔ ﺛﻼث ﻣﻨﻬﻢ ﺗﺤﺖ رﺗﺒﺔ
ﻣﺮآﺒﺎت اﻟﺘﻴﺮﺑﻮﻧﻴﺪ اﻟﺤﻠﻘﻴﺔ اﻟﺨﻤﺎﺳﻴﺔ وهﻢ ﺣﻤﺾ اﻟﻤﺎﺳﻠﻴﻚ واﻻﺳﻴﺴﺘﻚ واﻟﻌﺮﺟﻮﻧﻚ ﺟﻼﻳﻜﻮﺳﻴﺪك.
وﻋُﺮف ﻓﻲ ﺷﻜﻞ ﺳﻴﺲ وﺗﺮاﻧﺲ ارﺳﻔﺮاﺗﺮول -3-اوآﺴﻲ ﺑﻴﺘﺎ ﺟﻼﻳﻮل ﺟﻼﻳﻜﻮﺳﻴﺪ وﻋُﺮف
اﻟﻔﻼﻓﻮﻧﻴﺪ ﻓﻲ ﻣﺴﺘﺨﻠﺺ ﺧﻼت اﻻﻳﺜﻴﻞ ﺑﺨﺸﺐ اﻟﺸﺎف وﺗﺘﻀﻤﻨﺖ ﻣﺸﺘﻘﺎت اﻟﻜﻮارﺳﺘﻴﻦ ﺗﺤﺪﻳﺪًا
آﻮارﺳﺘﻴﻦ -7-اوآﺴﻲ ﺑﻴﺘﺎ داي ﺟﻼﻳﻜﻮﺳﻴﺪ و آﻮارﺳﺘﻴﻦ -7-اوآﺴﻲ .ﺟﻼﻳﻮل ﺟﻼﻳﻜﻮﺳﻴﺪ .ﺗﻢ
ﺗﺤﺪﻳﺪ ﻧﺎرﺟﻨﻴﻦ 4ﺑﺮاﻳﻢ ﻣﻴﺜﻮآﺴﻲ -7 -ارﺑﻨﻮﺳﻴﺪ ﻣﻊ ﻧﺎرﺟﻨﻴﻦ -7-ﺣﻤﺾ اﻻﻻﺟﻚ ﻓﻲ هﺬا
اﻟﻤﺴﺘﺨﻠﺺ ﺑﺎﻻﺿﺎﻓﺔ اﻟﻲ ، 6،5داي هﻴﺪروآﺴﻲ 3ﺑﺮاﻳﻢ 4ﺑﺮاﻳﻢ -7-ﺗﺮاي ﻣﻴﺜﻮآﺴﻲ ﻓﻼﻓﻮﻧﻮن
.
هﺬﻩ اﻟﻨﺘﺎﺋﺞ ﺗﺪﻋَﻢ اﻟﻬﺪف اﻟﻨﻬﺎﺋﻲ ﻻﺳﺘﻌﻤﺎﻻت ﻣﺴﺘﺨﻠﺼﺎت ﺧﺸﺐ وﻟﺤﺎء اﻟﺸﺎف ﻓﻲ اﻟﻤﻜﺎﻓﺤﺔ
اﻟﺤﻴﻮﻳﺔ ﺿَﺪ اﻟﻜﺎﺋﻨﺎت اﻟﻨﺒﺎﺗﻴﺔ اﻟﻤﻤﺮﺿﺔ .
xi
CHAPTER ONE
INTRODUCTION
Many of the plant materials used in traditional medicine are
readily available in rural areas and this has made traditional
medicine relatively cheaper than modern medicine (Mann, et.al.,
2008). Medicinal properties of plants are normally dependent on
the presence of certain phytochemical principles such as alkaloids,
anthraquinones,
cardiac
glycosides,
saponins,
tannins
and
polyphenols which are the bioactive bases responsible for the
antimicrobial property (Mann, et.al., 2008).
Secondary metabolites are a source of new antimicrobial products
and inexpensive starting materials for synthesis of many known
medicine, insecticide, fungicide and drugs etc. Considering the
great number of chemicals that have been derived from plants as
antimicrobial
compounds, scientific evaluation of plants used
traditionally for the treatment of some diseases or that possess
natural resistance to insect and fungi attacks seems to be logical
step of utilizing the antifungal compound, which may be present in
plants. Plants based antimicrobial compounds represent a vast
untapped source of medicine and pesticide with great potential
(Angeh, et.al., 2006). The use of chemicals to protect plants have
an advantage of being quick and effective, however, there are
disadvantages like toxic residues, extensive labour, pathogens
develop resistance to chemicals and chemicals also kill natural
enemies of pathogens (Eltahir, 2003). Consequently, the use of
chemical protectants in forest tree diseases control has not been
great (Rich, 1975).
1
Phytochemical analysis on Terminalia spp. (Combretaceae) started
since early 1970s and extended well into present. Important
secondary metabolites reported include, stilbenes, phenantherenes,
terpenoids, flavonoids and tannins, (Conrad, et al., 1998). The
cancer cell line active components were found to be gallic acid,
ethyl gallate, and the flavone luteolin. Only Gallic acid was
previously known to occur in this plant. Luteolin has a wellestablished record of inhibiting various cancer cell lines and may
account for most of the rationale underlying the use of T. arjuna in
traditional cancer treatments. Luteolin was also found to exhibit
specific activity against the pathogenic bacterium (Conrad, et al.,
1998). T. stuhlmannii stem and bark yielded two glycosides of
hydroxyimberbic acid, one of which is reported for the first time,
associated activities with these compounds are anticancer,
antimalarial, anti-inflammatory, gastroprotective and antimicrobial
activities. Bioassay-guided separation methods, led to the study of
cancer cell growth inhibitory constituents residing in the bark, stem
and leaves of Terminalia spp. The structure of the isolated
compounds was elucidated by spectroscopic methods. Several
compounds had antibacterial activity, imberbic acid showing
particularly potent activity against Muycobacterium fortuitum and
staphylococcus aureus. (http://www.sciencedirect.com. 2007).
Among the reasons for pursuing natural product chemistry resides
in the actual or potential antimicrobial activity to be found in
alkaloids, terpenoids, coumarins, flavonoid, lignans and other
secondary metabolites. The use of plants derivatives as a source of
antimicrobials has been virtually non-existent since the advent of
antibiotics in the 1950s (Angeh, 2006).
2
Secondary metabolites content varies among wood species,
between bark and wood of the same species. Differences between
the species may be anticipated on the basis of differences in
chemical composition. Abdelhameed (2003) found substantial
differences between leaves, bark, wood, roots and flowers.
Terminalia brownii is distributed in wide range of Savanna zones
on loamy soil in Darfor, Kordofan, Blue Nile, Kassala and South
Sudan. T. brownii is one of the most resistant plant species to
many pathogenic fungi that affects the savannah forests in those
areas. Secondary metabolites found in the different parts of this
plant was suggested to be responsible for its resistance i.e. T.
brownii different extracts were expected to have antimicrobial
activity.
1.1 Objectives of this study
To date no phytochemical studies are reported on T. brownii. The
main objective of this study is to chromatographically profile and
biologically screen T. brownii (wood and bark) extracts,
elaborating their antimicrobial activity, isolating their active
ingredients and elucidating their structure. The objectives could be
achieved by:
• Biologically guided fraction of the different extracts
obtained with special emphasis of their antimicrobial
activity.
• Separation of the active fraction using high performance
liquid chromatography (DAD. HPLC).
• On line identification of the major compounds residing in
the active fractions using Tandem mass spectroscopy (LC
MS MS).
3
CHAPTER TWO
LITERATURE REVIEW
2.1 Taxonomy of the genus Terminalia
Terminalias are a medium sized to large trees up to 20 m high.
Bark grayish white, becoming very dark grey, scaly in old trees.
Branches often drooping and slender. Leaves alternate, rarely
opposite or subopposite; elliptic to ovate- lanceolate, 2 – 8 x 1.3 –
5 cm; densely silky becoming pubescent beneath. Inflorescence
small, greenish-yellow globose head; petals absent. Fruits 2
winged fruit in globose or subglobose cone-like heads,
coriaceous, broadly winged dark grey. Flowers in globose heads,
small, greenish yellow, (Elamin, 1990). According to Carolus
linnaeus the plant studied could be classified as: -
Kingdom:
Subkingdom:
Plantae: Plants
Tracheobionta -- Vascular plants
Superdivision: Spermatophyta -- Seed plants
Division:
Magnoliophyta -- Flowering plants
Class:
Magnoliopsida -- Dicotyledons
Subclass:
Rosidae
Order:
Myrtales
Family:
Combretaceae
Genus:
Terminalia L.
Species:
brownii
S.N:
Terminalia brownii
Vernacular name:
Al shaf, Al drot, Alsafraya, Alsobag
4
2.2 Antimicrobial metabolites of plant origin
Plants have almost limitless ability to synthesize different types of
secondary
metabolites.
Useful
phytochemicals
that
have
antimicrobial effects can be divided into several categories these
include, simple phenols and phenolic acid e.g. cinnamic and caffeic
acid which are effective against viruses, bacteria and fungi, (Cown,
1999). In addition more investigations revealed that terpenes are
toxic for fungi and for bark beetles (Conrad, et.al., 1998).
Contribution of secondary metabolites as antimicrobial activity
also refer to active constituent namely triterpens and saponin like
mollic acid, jessic acid and their derivatives, the sodium salt of
mollic acids glycoside isolates from C. molle were found be toxic
to Biomphalaria glabrata snails (Angeh, 2007). More recently
series of stilbenes and dihydrostilbenes (combretastatin) with
potent cytotoxic activity and acidictriterpenoids and their
glycosides with molluscicidal, antifungal and anti-inflammatory
activity have been isolated from Terminalia species
Some essential oils are effective against some higher organisms
such as nematodes, helminthes and insects. Common active
components of the essential oils include thymol, carvacol, camphor
and terpinene-4-ol (Ncube, et. al., 2007). Many of the woods from
which stilbenoids have been isolated are highly resistance to decay
Stilbenes are involved in the protection of wood decay and are
induced
as
phytoalexins
(phenanthrenes
and
dihydro-
phenantherenes) in response to pathogenic attacks (Seigler, 1998).
5
2.3 Combretaceae secondary metabolites and their biological
significance
Species of Combretaceae contain compounds with potential
antimicrobial properties (Angeh, 2006). There is a large variation
in the chemical composition and antimicrobial activity among the
different genera and species in the Combretaceae. Several species
of Combretaceae used in traditional medicine in West Africa have
been investigated for their antifungal activity against the
pathogenic fungi. Phytochemistry screening revealed that these
plants are particularly rich in tannins, and saponins, which might
be responsible for their anti-fungal activity (Baba-Moussa, et.al.,
1999).
Stilbene aglycone are common in heartwood, living tissue often
contents small amount of stilbenes glycoside (Figure 1-A), many of
the wood from which stilbenoids have been isolated are highly
resistance to decay (Seigler, 1998).
To date around 80 metabolites were reported from the genus
combretum
including
stilbenes
phenantherenes,
terpenoids,
cycloarenoids, macrolactones and flavonoids (Pettit, et.al., 1995;
Jossang, et al., 1996; Adnyana, et.al., 2000, Ogan, 1972;
Abdurzag, et.al., 1997). One of the most active stilbenes isolated is
combretastatin A4, which is in very late stages of clinical trials.
2.4 Terminalia important secondary metabolites
Phytochemical work on Terminalia spp. started since early 1970s
and extended well into present. Important secondary metabolites
reported include, stilbenes, phenantherenes, terpenoids, flavonoids
and tannins, (Conrad, et.al., 1998). Associated activities with these
6
compounds
are
anticancer,
antimalarial,
anti-inflammatory,
gastroprotective and antimicrobial activities. By means of
bioassay-guided separation methods, the cancer cell growth
inhibitory constituents residing in the bark, stem and leaves of the
Mauritius medicinal plant Terminalia arjuna (Combretaceae) were
examined. The cancer cell line active components were found to be
gallic acid, ethyl gallate, and the flavone luteolin. Only gallic acid
was previously known to occur in this plant. Luteolin has a wellestablished record of inhibiting various cancer cell lines and may
account for most of the rationale underlying the use of T. arjuna in
traditional cancer treatments (http://www.sciencedirect.com. 2008).
Luteolin was also found to exhibit specific activity against the
pathogenic bacterium. Terminalia stuhlmannii stem and bark
yielded two glycosides of hydroxyl imberbic acid, one of which is
reported for the first time. The structure of the isolated compounds
was elucidated by spectroscopic methods. Several compounds had
antibacterial activity, imberbic acid showing particularly potent
activity Against Muycobacterium fortuitum and staphylococcus
aureus (http://www.sciencedirect.com. 2008).
The root and bark of Terminalia sericeae yielded an unreported
stilbene
glycoside,
3'5'-dihydroxy-4-
(2-hydroxy-ethoxy)
resveratrol-3-O-β-rutinoside together with known compounds
resveratrol-3-β-rutinoside glycoside, 3', 4,5’-Trihydroxystilbene
(resveratrol) as shown in figure 1-A. Structure determination of the
isolated compounds was achieved on the basis of spectroscopic
measurements (Joseph, et.,al., 2007).
Many biologically active compounds were detected and their
structures were elucidated, from the genus Terminilia, this
includes:
7
1,3-diarylpropanes,1-(4'-hydroxy-2'-methoxyphenyl)-3-(3"methoxy
4"hydroxyphenyl)-propane
dimethoxyphenyl)-3-(3"-methoxy-4"
seven
flavanones,
sakuranetin,
naringenin,
isosakuranetin,
liquiritigenin-7-methyl-ether
two
chalcones,
and
1-(2'-hydroxy-4',6'-
-hydroxyphenyl)-propane,
naringenin-4',7-dimethyl-ether,
liquiritigenin-4',7-dimethyl-ether,
and
liquiritigenin-4'-methyl-ether,
isoliquiritigenin-4-methyl-ethe
and
isoliquiritigenin-4-methyl-ether, one flavan, 7,4'-dihydroxy-3'methoxyflavan (Figure 1-B), nine triterpenes (Figure 1-C), arjunic
acid, arjunetin, arjungenin, arjunglucoside I, arjunolic acid,
arjunglucoside II, 23-galloylarjunglucoside II (isolated as its mono, di- and tri-O-methyl derivatives after methylation with
diazomethane), betulinic acid and ursolic acid acetate, along with
gallic acid and sitosterol were isolated from the heartwood and
bark of Terminalia fagifolia (http://www.scielob. 2008).
A new oleanane-type triterpene (3β, 6β, 23, 28-tetrahydroxyolean12-ene) was isolated from the leaves of Terminalia glabrescens,
together with ursolic, 2α-hydroxyursolic, oleanolic, maslinic,
arjunolic, sumaresinolic and asiatic acids, squalene, phytol,
sitosterol-3-O- β -D-glucopyranoside and n-alkanes. Friedelin,
taraxerol, lupeol, lupenone, betulin, betulone, betulinic acid,
stigmastane-3 β, 6 α -diol, - β sitosterol, catechin, β-Dpyranotagatose, β-D-furanofructose and α-D-furanofructose were
obtained from the trunk bark of T. glabrescens (www.scielo.br,
2008). A new cardenolide, 16,17-dihydroneridienone 3-O-β-Dglucopyranosyl- (1→6)-O-β-D-galactopyranoside, was isolated
from the roots of Terminalia arjuna. (Yadav, et.al., 2000). T.
calcicola led to the isolation of two new cytotoxic xanthones,
termicalcicolanone A and termicalcicolanone B. Both compounds
8
showed modest antiproliferative activity toward the human ovarian
cancer cell line (Cao, et.al., 2008).
9
OH
OH
HO
H
HO
OH
H
O
OH
HOHO
OH
Resveratrol
T, sericeae
Revesatrol -beta-D-glycoside
T. sericeae
OH
H
MeO
HO
H
H
MeO
OMe
H
OH
OMe
OH
O
O
O
H3C
HO
Combretastatin A4
Combretum caffrum
OH
OH
OH
Reveratrol-3-ortho-beta-rutinoside
T.sericeae
H
OH
HO
H
OH
OH
O
O
O
H3C
HO
OH
OH
OH
Stilben glycoside
T.sericeae
Figure 1.A: Stilbens reported in Combretaceae
10
OCH3
OCH3
OH
OH
Diarylpropane
Teminalia spp.
OH
Diarylpropane
Terminalia spp.
OCH3
OCH3
H3CO
H
HO
OCH3
OH
OH 3C
HO
O
O
OH
O
O
OH
,
Isosakurantin(Naringenin-4 -methyl ether)
T.fagifolia
Sakuranetin
T. fagifolia
OH
OH
OH
OH
OH
O
OH
O
OH
O
Isoliquiritigenin
T. fagifolia
Luteolin
T. arjuna.
OCH3
OCH3
OH
OH
HO
O
H
O
,
Isoliquiritigenin-4 -methyl ether (2,4
,
dihydroxy-4 -methoxy chalcone)
T. fagifolia
O
Liquiritigenin-4,-methyl ether
T. fagifolia
Figure 1.B.1: Flavonoids reported in Terminalia spp
11
OH
O
OH
O
OCH3
OH
O
OH
7,4, dihydroxy-3,-methoxy falvan
T. fagifolia and T.aregentena
Liquiritigenin
T. fagifolia
CH3O
HO
O
OH3C
OH
O
O
H
OH
O
Liquiritigenin-4,,7-dimethyl ether
(7,4,-dimethoxy flavan)
T. fagifolia
Genistien
T. arjuna
OH
CH3O
OH3C
OH3C
O
O
H
OH
O
O
,
liquiritigenin-7-methyl ether
T.fagifolia
Narigenin-4 ,7dimethyl ether
T. fagifolia
OH
OH
OH
HO
HO
O
O
OH
O
OH
OH
O
Naringenin
T. fagifolia
Quercetin
T. arjuna
Figure 1.B.2: Flavonoids reported in Terminalia spp continued
12
H 3C
H 3C
CH3
H
H
O
O
O
O
OH
OH
O
CH3
O
O
OH
O
OH
T erm icalcicolanon e A
T . calcicola
T erm icalcicolanon e B
T . C alcicola
Figure 1.B.3: Flavonoids reported in Terminalia spp continued
13
HO
OH
C H 2 OH
O
OH
OH
O
HO
C H 2 OH
CH 2 OH OH
OH
OH
3b eta,6beta,23,28 tetrah y d roxy olean -12-ene
T . glabrescens
OH
A rju n ic gly cosid e 1
T . arjuna T .fagifolia
HO
C OO Gl u
OH
C OO H
HO
OH
O
HO
O
CH3
A rju n ic acid
T . arjuna T . fagifolia
H
H
OC H 3
23-(3,4-d i-orth o-m eth y l) galloy l arju n
T . fagifolia T . m acropetra
H
OH
O
H
CH3
HO
S q ualen e
T . glabresces
OH
S u m arson lic acid
T erm inalia spp
HO
OH
CO
HO
C OO H
O
O
HO
CH3
A rju n etin
T . arjuna T .fagifolia
OH
OH
HO
OH
OH
C H 2 OH
OH
Figure 1.C.1: Terpenes reported in Terminalia spp
14
A rju n gen in
T . arjuna
H
H
OH
H
H
H
H
H
HO
H
HO
Sitostrol
T. glabrescen
Betulin
T. glabrescens
H3C
COOH
CH3
OH
H
H3C
H
H
H3C
H
CH3
HO
CH2OH
CH3
Imberbic acid
T.stuhlmanii
O
CH3
Friedelin
T. glabrscens
CH3
CH3
H2C
H
H
CH3
CH3
H
CH3
H
CH3
H
H
H
OH
H3C
HO
H
CH3
Lupeol
T. glabrescens
Stigmasterol
Terminalia spp.
CH3
O
O
CH3
CH3
H
H
H
H
CH3
H
CH3
H
H3C
H
H
Caredenolide
T.arjuna
OH
CH3
Terminalin A
T. glaucescens
Figure 1.C.2: Terpenes reported in Terminalia spp continued
15
CH3
In Terminalia macroptera and Terminalia belerica, previous
investigations have demonstrated that the leaves content of
secondary metabolites changes after artificial inoculation with
fungi. These modifications vary according to the resistance level of
Terminalia spp. The study of protective responses of Terminalia
spp to attack by pathogenic fungi is essential for developing new
approaches to assess the resistance of trees to infectious diseases.
In some cases, the rate of lignin accumulation can characterize the
tolerance of a plant towards both fungal pathogens and for the
insect.
Medicinally the barks are used for cough and bronchists and
fumigant is used for rheumatism (El-Ghazali, et.al., 1997). The
local knowledge in using species in body fumigation, cosmetic and
folk medicine such as inflammatory and jaundice are well known
to the forests-adjacent communities. Other Terminlia spp in Sudan
and Africa and their traditional uses are summerized in Table 1
16
Table: 1 African and Sudanese traditional uses of Terminalia spp
Terminalia chebula (in Sudan)
Terminalia fatraea
Part
used
Leaf
Root
Leaf
Latex
Root
Bark
Root
Bark
Root
Bark
Leaves
Fruit
Fruit
Bark
Diarrhea
Conjunctivitis, phagedenic ulcer, wounds
Rheumatism
Conjunctivitis, ear inflammation
Diarrhea
Diarrhea, stomach pain
Hematuria, cholemesis
Jaundice, malaria
Cough
Dysmenorrhagia, jaundice, yellow fever
Hypertension, diabetes
Sever diarrhea
Asthma, cough, hypertension Chronic ulcer, laxative
Colic, indigestion
Terminalia glaucescens
Leaf
Burns, headache, stomach pain, cough,
Terminalia arjuna (in Sudan)
Root
Leaves
Root
Bark
Bark
Leaves
Root
Bark
Leaves
Bark
Root
Leaves
Root
Bark
Leaves
Root
Bark
Bark
Root
Bark
Root
Bark
Terminalia brownii (in Sudan)
Bark
Dental care
Wounds, hemostatic,hemorrhoids.malaria, yellow fever
Kidney pain, cough, headache,
Asthma, cancer,
Skin pustules, wounds, hemostatichemorrhoids
Hemorrhoids
Jaundice,
Wound, hemostatic, hemorrhoids
Severe diarrhea
Polymenorrea, cholera
Gastrointestinal disorders, cougbronchitis
Diarrhea, stomach Dysentery, colic,
Wounds, diarrhea, skindisease,cough,stomache
Diabetes, diarrhea, dysentery, colic
Malaria
Epilepsy
Diuretic,insanity,cholemesis,dysentery,vomiting
Abdominal pain
Stomach pain
Bloody diarrhea cancer, gastric ulcers
Cancer, gastric ulcers
Astringent, tonic, febrifuge, diarrhea
Antinflammatory, cough bronchists
Bark
Fumigant for rheumatism and cosmetics,
Fruit
Expectorant, antiseptic
Species
Terminalia albida
Terminalia avicennioids
Terminalia basilie
Terminalia bauman
Terminalia brachystemma
Terminalia brevipes
Terminalia brownii
Terminalia catappa
Terminalia ivorensis
Terminalia kaiserana
Terminaliakilimandscharica
Teminalia laxiflora
Terminalia macrocarpa
Terminalia mollis
Terminalia monoceras
Terminalia orbicularis
Terminalia prunioides
Terminalia sericea
Terminalia spinosa
Terminalia stenostachya
Terminalia superba
Terminalia trichopoda
Terminalia zambesiaca
Terminalia belerica (in Sudan)
Claimed therapeutic uses
Neuwinger, (2000), Ahmed, et.al., (1998), El-Ghazali, et.al., (1997)
17
2.5 Plant pathogen tested
Aspergillus
niger
(ATCC9763-8/29/2005)
(aerobic
fungi
Eurotiomycetes) belongs to the class Eurotiomycetes order
Eurotiales are filamentous fungi common in the environment. Asp.
niger can cause the rotting of numerous fruits, vegetables, and
other food products. A. niger causes black mould of onions.
Infection of onion seedlings by A. niger can become systemic,
manifested only when appropriate conditions are available. A.
niger causes a common postharvest disease of onions, in which the
black conidia can be observed between the scales of the bulb. The
fungus also can infect in peanuts and grapes. A. niger damages
surface layers of wood, raw cotton fibers and many other materials
it is used to test the efficacy of preservative treatments (Jong, and
Gantt, 1987). A. niger is a common laboratory contaminant and can
causes disease of mycotoxins, where systemic infection is often
fatal. (http://www.en.wikipedia.org/wiki/ 2008).
Nattrassia.
mangiferae
(Eltahir,
2003)
(Imperfect
fungi,
Deuteromycetes) belongs to the class Coellomycetes order
Sphaerospidales causing cankering of the main stem, wilting of the
branches is associated with decline of trees. (Eltahir, 2003).
Nattrassia mangiferae is a common fungus that causes branch wilt
in Sudan. It has a wide host range consisting of fruit trees, shade
trees, and ornamental trees. N. mangiferae infection causes about
33.3-95.34% and 79.16-100% mortality in Ficus nitida and Ficus
benjamina, respectively. The first infection by N. mangiferae on
humans had been reported in India in 1970. It causes dry, scaling
skin disease. Infection of humans is thought to occur by contact
with infected soil, although in some cases people become infected
by direct contact with splinters of wood. Human infection by this
18
fungus is probably most common than is reported, because it could
easily be mistaken with other diseases. Infection of humans usually
occurs in tropical and subtropical areas where the fungus is
endemic (Eltahir, 2003).
Aspergillus
flavus
(N.H.L,
2006)
(aerobic
mould
fungi
Eurotiomycetes) belongs to the class Eurotiomycetes order
Eurotiales. The mold damage A. flavus is one of the most
important causative agent of damage for corn and peanuts, and it is
the one of several species of moulds known to produce aflatoxin.
Aflatoxin in human causes acute hepatitis, immuno-suppression,
and hepatocellular carcinoma. (http://www.wikipedia.odjechane.
2008). It is a pathogen, associated with aspergillosis of the lungs
and sometimes believed to cause Corneal, Otomycotic, and
nasoorbital infections. (http://www.wikipedia.odjechane. 2008).
Fusarium moniliforme (PPI/PPA/MA, 2006) (filamentous fungus
Hypocreaceae) belong to the class Sordariomycetes order
Hupocreales, it is fungus infecting soybean, bean and other crops.
It causes bakanae disease in rice seedlings, by overloading them
with
the
phytohoromone,
gibberellins
(http://www.wikipediaodjechane.net. 2008). Southern forest pine
heavily infected with Fusarium species are predisposed to be killed
by the Cankers (George, 2004). The inflorescences of Mango
(Mangifera indica) when attacked by Fusarium moniliform are
commonly followed by other fungi like Aspregillus niger. Fungal
mycelia gradually invade the xylem tissues from the top of the
branches and spread basipetally ultimately causing death of the
infected branches (Rajput, and Rao, 2004).
19
2.6 Biosynthesis of Flavonoids
Flavonoids are member of a class of natural compounds
(phenylpropanoids) with widespread occurrence in plant kingdom.
Plant phenols are being regarded as those substances derived from
the shikimate pathway and phenyl propanoid metabolism. The
flavonoids are built upon a C6 -C3-C6 flavones skeleton in which
the three-carbon bridge between the phenyl groups is commonly
cyclized with oxygen (Robards, and Antolovich, 1997). The
biosynthesis of flavonoids compounds are derived from a branch of
the flavonoid pathway (Figure 2), for which chalcone synthase
(CHS) provides the first committed step by condensing one
molecule of p-coumaroyl-CoA with three molecules of malonylCoA to produce tetrahydroxychalcone (a chalcone, Figure 2).
Chalcone provides the precursor for all classes of flavonoids,
which include the flavones, flavonols, flavan-diols, flavan 4- ols,
proanthocyanidins
(condensed
tannins),
isoflavonoids,
and
anthocyanins. The closure of the C-ring, resulting in the formation
of flavanones, is carried out by chalcone isomerase (CHI).
Flavanones (e.g., naringenin) provide a central branch point in the
flavonoid pathway and can serve as substrates for enzymes that
introduce–OH groups at the 3' and 5' positions of the B-ring, or for
the hydroxylation of the C-ring by flavanone 3-hydroxylase.
Dihydroflavonol 4-reductase (DFR) provides one entry step to the
biosynthesis of anthocyanins, and depending on the plant species, it
can utilize as a substrate any one or all three of the possible
dihydroflavonols,
dihydromyricetin,
dyhydroquercetin.
20
dihydrokaempferol,
or
The leucoanthocyanidins are converted into the corresponding
anthocyanidins
by
the
action
of
a
leucoanthocyanidin
dioxygenase/anthocyanidin synthase. (Grotewold, 2006)
21
OH
SCoA
P-Coumaroyl
O
HO
OH
Chalcones
OH
O
OH
TetrahydroxyChalcone
OH
OH
B
OH
HO
O
O
OH
Pentahydroxyflavanone
OH
OH
OH
OH
HO
3Malonyl-CoA
+
A
C
OH
O
O
Naringenin
OH
O
O
Flavanones
OH
OH
O
HO
O
O
O
OH
OH
Dihydromyricetin
OH
OH
OH
OH
OH
OH
OH
OH
HO
Eriodic lyol
OH
O
Dihydrokaempferol
O
Dihydroque rcetin
Dihydroflavonols
Leucodelphinidin
Leucopelargonidin
Leucocyanidin
Luteoforol
Leucoaanthocyanindins
Delphindin
Pelargonidin
Cyanidin
Anthocyanidins
Figure 2: Biosynthesis of the different classes of flavonoids
22
Luteolindin
2.7 Chromatographic analysis of flavonoids
In both profiling and quantification studies of flavonoids, the most
successful approaches to date have been based on chromatography.
Thin layer chromatography is an ideal technique for the screening
of antimicrobial activity because of it is low cost, easy maintenance
and selectivity of detection reagents. TLC on silica gel is very
favorable for the analysis of flavonoids, (Stobiecki, 2000, Robard
and Antolovich, 1997). The selection of the suitable stationary
phase and solvent depend on the class of flavonoids to be
examined.
High performance liquid chromatography (HPLC) and Gas liquid
chromatography (GLC) are important where identification is
required. Liquid chromatography (LC) of flavonoid is usually
carried out in the reversed-phases (RP) mode. On C8-or C18bonded silica columns. Gradient elution is generally performed
with binary solvent systems, i.e. with water containing acetate or
formate buffer and methanol or acetonitrile as organic modifier.
LC is usually performed at room temperature, but temperature up
to 40° C are sometimes recommended to reduce the time of
analysis and because thermostated columns give more repeatable
elution times. If the main aim of the study is to determine the major
flavonoids in a sample, run times of 0.5-1 h usually suffice to
separate five to ten compounds of interest (Rijke, et.al., 2006).
Flavonoid detection is carried out at 250, 265, 290, 350, 370, and
or 400nm (with an added wavelength in 500-525nm ranges if
anthocyanidins are included. (Rijke, et.al., 2006).
23
2.8 Stilbenes and their biosynthetic pathway
There are two major groups of the stilbenes, resveratrol (stilbenes),
and the phenanthrenes, together with their respective dihydro
derivatives are characteristics in Combretaceae family (Seigler,
1998).
The stilbenes are often in plants that are not routinely consumed for
food or in the nonedible tissue (Cassidy, et.al., 2000), and are
usually assumed that the resistance of these woods to fungal attack
is due to presence of these phenolic materials. Other higher plant
source includes Combretaceae (Harborne, et.al., 1999). Stilbenoids
are widely distributed in higher plants, 29 in monomeric form and
as dimeric, trimeric and polymeric stilbenes, the so-called
viniferins. Among monomeric stilbenes, trans-resveratrol has been
identified as the major active compound, and most of the studies in
the literature about the physiological activity have focused on it;
however, there are also some studies of the 3-β-glucoside of
transresveratrol, the so-called piceid or polydatin, and the
viniferins. (Cassidy, et.al., 2000).
Phenanthrenes are rather uncommon class on aromatic metabolites,
where as presumably formed by oxidative coupling of the aromatic
rings of stilbene precursors. Besides these stilbene derived
compounds, phenanthrenes most likely originated from diterpenoid
precursors
(Cassidy,
2000).
et.al.,
Biosynthesis
of
dihydrophenanherenes is similar to that of stilbenes, but appears to
involve dihydrocinnamic acids and the enzyme bibenzyl synthase,
where as the biosynthesis of phenantherenes involves the
corresponding unsaturated acids (Seigler, 1998).
24
The
phenanthrenes
Monophenanthrenes,
classified
into
diphenanthrenes
three
and
major
groups.
triphenanthrenes.
Monophenanthrene are subdivided according to the number and
type of the structural moieties, while the type of connection of the
phenanthrene
units
can
classify
diphenanthrene,
Tricyclic
9,10dihydrophenanthren originate from phenylpropane derivatives
by chain elongation and cyclization according to the polyacetate
rule. Bibenzyls are bicyclic intermediates, and O-methylation is a
prerequisite for their conversion into dihydrophenanthrenes.
(Preisigmuller, et.al., 1995). Up to present only one compound of
the triphenanthrene group was described. (Kovacs, et.al., 2007).
Large number of biological activities of differently substituted
phenanthrene has been reported to occur in plants and has been
demonstrated to possess various active compounds, Phenanthrenes
have been studied for their cytotoxicity, antimicrobial, spasmolytic,
anti-inflammatory, antiplatelet aggregation, antiallergic activities
and phytotoxicity, most natural phenanthrenes occur in monomeric
form, this group consist about 210 compounds, almost 100 are only
hydroxy- and/or methoxy- substituted, and equally 9, 10-dihydro or
dehydro-derivatives. Their great structural diversity stems from the
number and position of their oxygen functions. The hydroxyl and
methoxy moieties number are between 3 and 6, and can usually be
found on C-2, C-3, C-5, and C-6 or C-7. Besides hydroxyl and
methoxy groups, further substituents can be found in monomeric
phenanthrenes, such as methyl, hydroxylmethyl, carboxy, formyl,
prenyl and vinyl. Another type of monomeric phenanthrenes is the
group of phenanthraquinones; altogether 19 compounds belong to
this group. They are usually hydroxyls, methoxy or methyl
substituted (Kovacs, et.al., 2007).
25
Stilbenes are 1, 2-diarylethenes (Figure 3), Ring A usually carries
two hydroxyl groups, while ring B is substituted by hydroxy and
methoxy groups in the 0-, m- and/or p-position. The stilbenoids are
group of phenolic compound biosynthetically inter related through
their common origin from a C6-C2-C6 intermediate. Like
flavonoids, they are formed from the condensation of a Phydroxycinnamic acid (C6-C3) precursor with three molecules of
malonyl coenzyme A, but they differ in that one carbon atom is lost
by (decarboxylation) in the process. Biosynthetic activities require
4-coumaroyl-CoA and three malonyl-CoA; these are present in all
plants (Harborne, et. al., 1999). The reactions of resveratrol
synthase and chalcone synthase are very similar, and only the final
ring-folding is different in resveratrol synthase. Resveratrol
synthase and chalcone synthase are condensing enzymes; they use
three sequential condensation reactions with malonyl-CoA to
produce an enzyme-bound tetraketide intermediate (Harborne, et.
al., 1999).
26
OH
SCoA
O
4-coymaroyl-CoA
Malonyl-CoA
COASH
OH
CO2
SCoA
O
O
Diketide
Malonyl-CoA
COASH
OH
CO2
SCoA
O
O
O
Triketide
Malonyl-CoA
COASH
OH
CO2
O
O
O
O
Tetraketide
CO2
CoASH
Molonyl-CoA
OH
B
OH
COASH
A
CoASH CO2
OH
OH
OH
OH
O
Chalcone
Figure 3: Stilbenes biosynthesis (Schroder, 1990)
27
Reseveratrol
2.9 Chromatographic analysis of stilbenes and phenantherenes
Stilbenes and related compounds can be separated with a number
of techniques such as thin-layer (TLC), gas liquid chromatography
(GLC), and high performance liquid (HPLC) chromatography,
characterization and identification of these compounds can best be
accomplished by nuclear magnetic resonance (NMR) spectroscopy
and mass spectroscopy (Seigler, 1998).
To investigate the presence of stilbenes in red wine a new reversedphase (RP) high-performance liquid-chromatographic (HPLC)
method with enhanced separation efficiency and improved
selectivity, sensitivity, and speed has been established for
determination of the stilbenes cis- and trans-resveratrol, in a single
run. UV-absorbance, fluorescence (FLD), and mass-spectrometric
(MS) detection were also evaluated. UV-absorbance detection was
adjusted at 320 nm for stilbenes (Laszlo, et.al., 2005).
To quantify and qualify phenantherenes in biological matrices,
detection and identification of the anylte (phenantherenes) were
achieved using gas chromatography coupled to mass spectroscopy
(Grova, et.al., 2005). RP-HPLC, column, elution systems of mobile
phase and the detectors effected the separation of phenantherenes
(Arbabi, et.al., 2004). In the other case phenanthrene concentration
in contaminant soil was measured by HPLC, with chromatographic
conditions as follows: Analytical column C18 Ultra Sep ES PAH
QC Speica, 60 × 2 mm ID. Flow rate (ml/min) 0.5, injection rate 50
µl, Elute acetionitirl/water: 40-100%, UV detector wavelength: 254
nm according to pre-test results the optimum elute condition for
phenanthrene was determined at 60/40 (acetonitril/water) (Arbabi,
et.al., 2004)
28
CHAPTER THREE
MATERIALS AND METHODS
3.1 Plant material collection
The plant material (wood and bark) used in this study was collected
from El Nour Natural Forest Reserve, southeast of El Damazine
district-Sudan. Geographically located between latitude 11˚ 52.5`
and 11˚ 48` N; longitude 34˚ 30`and 34˚ 29.5` E. Wood and bark
pieces were collected separately from Terminallia brownii
(Combretaceae) trees free from diseases, accumulation of knots,
resin galls and gums. A voucher specimen was made for the plant
studied and identified by taxonomist in the department of
siliviculture, Faculty of Forestry, University of Khartoum. The
herbarium was deposited at the Department of Biochemistry,
Commission of Biotechnology and Genetic Engineering, National
Center for Research. Collection data categorized under place, date
and collector, an asterisk indicating a herbarium sample are as
follows: Terminallia brownii (1*), El Nour Natural Forest Reserve,
May – 2006, Enass, (Figure 4).
29
A
B
C
D
Figure 4: Terminalia brownii parts
A. Whole Plant
B. Bark
C. Leaves
D. Inflorescence
30
3.2 Plant material preparations and extractions
Samples were taken according to Koch (1985) from healthy old
trees growing in that natural forest 50 cm long logs (diameter 2042 cm) above 180cm from the ground All logs were manually
debarked; samples were air dried under shade. The wood and barks
samples were chipped to small chips using sawmill followed by
hammer mill (mesh.) to obtain finely grounded wood powder
which were stored separately in paper bags. Hundred grams of the
air dried species were extracted sequentially using solvent of
increasing polarities. The plant material was extracted using
petroleum ether (PE), marc was then extracted with chloroform
(Ch) and finally the marc was extracted using 80% methanol
(MeOH).
The
methanolic
extract
was
then
fractionated
(liquid/liquid) using ethyl acetate (EtoAc). Extracts were
concentrated to dryness by evaporating the solvent at room
temperature, (Figure 5).
31
100gm
Wood/ Bark
Petroleum
ether
PE extracts
Marc
Chloroform
CHCL3 extract
Marc
Aqueous MeOH %80
MeOH extract
Liquid/liquid
extraction (EtOAc)
EtOAc Fraction
Aqueous Part
Figure 5: Schematic diagram of T. brownii wood and bark
extraction
32
3.3 Chromatography
3.3.1 Thin Layer Chromatography (TLC) analysis for plant
extracts
Thin layer chromatography was carried out using silica gel plates
60F254 (Merck5554) or pre-coated TLC plates SIL RP-18W/UV
254 (Macherey-Nagel). Chromatograms of the plant extracts were
prepared by applying 20µl solution (5mg/ml) to the silica gel plate
and developing it in different solvent systems depending on the
type of the extract, (Table 2). Chromatograms were detected under
UV light UV lamp (Camag), (254 and 366nm) and sprayed with
diagnostic
reagents
which
include
vanillin-H2SO4reagent,
Aluminium chloride and Natural Products reagent.
33
Table 2: Developing Solvent systems used in TLC
Developing system
Ratio of Solvents
Methanol/ Water/ Acetic acid RP
8:1:1
Methanol/ Water/ Acetic acid RP
7:1:2
Methanol/ Water/ Acetic acid RP
6:2:2
Toluene / Ethyl acetate/ Formic acid NP
8:1:1
Toluene/ Ethyl acetate/Formic acid NP
5:4:1
Toluene/ Ethyl acetate/ Formic acid NP
4:5:1
34
3.3.1.1 Spray reagents
•
Vanillin H2SO4 : was prepared as follows: 1gm of
vanillin powder dissolved in 90ml methanol to which
10 ml sulphuric acid was added carefully. Sprayed TLC
plates were examined after heating at 120 0C.
• Dragendorff Reagent: Composed of two solutions:
- Solution A: 0.3 g bismuthsubnitrate in 1 ml of 25%
HCL and 5 ml H2O
- Solution B: 3 g potasium iodide in 5 ml H2O
The spray reagent was composed of 5 ml (A) + 5 ml
(B) + 5 ml of 12.5% HCL + 100 ml H2O.
• Natural
Products
(polyethylenglycol)(NP/PEG)
Reagent: Plates were sprayed with 1% methanolic
diphenylboric acid (NP), followed by 5% ethanolic
polyethyleneglycol – 4000 (PEG) (10 ml and 8 ml,
respectively).
3.4.2 Solid Phase Extraction (SPE)
Sorbents for SPE were LC-18 reversed phase packings supplied by
Supelco. Before applying the sample, the column was equilibrated
with the first designated eluent. For LC-18 silica the starting eluent
was 100 % H2O, 50% H2O: MeOH and the column was finally
washed with 100 % MeOH.
35
3.4.3 Reverse Phase High Performance Liquid
Chromatography (RP-HPLC)
The Agilent 1100 series HPLC system was composed of Agilent
series 1100 thermostated column compartment, Agilent series 1100
autosampler, binary Agilent 1200 series Bin pumps, Agilent series
1100 vacuum degasser and Agilent series 1100 DAD detector.
Plants extracts and fractions were separated on a (Varian LC-18,
4.6 mm x 250 mm x 5 µm USA) reverse-phase column at 30oC and
a flow rate of 0.5 mL/min. The column was eluted with a gradient
mobile phase consisting of 1% acetic acid in H2O (phase A) and
100% acetonitrile (Solvent B) using the gradient programs
presented in Table 1. UV detection was performed at 320-380 nm
for flavonoids and stilbenes, respectively.
3.4.4 LC-triple quadruple spectrometric analysis (LC-MS/MS)
The HPLC was joined with a HTC ultra-Bruker Daltonics Advanced Mass Spectrometry Instrumentation (Germany) with
Electrospray Ionization (ESI) interface at alternative ion mode. The
capillary temperature was set to 280oC and the spray voltage was
set to 5000 V. Nitrogen was used as sheath gas, and the flow was
set to 40 U. Helium was used as collision gas at 0.8 mTorr.
Collision Induced Dissociation (CID) or IT-MSn experiments were
performed for fragmentation of the compounds studied. Neutral
loss scan were investigated with scan range from m/z 50 to 1000 at
collision energy of 15 and 30 ev.
36
3.5 Antimicrobial activity
The extracts of T. brownii were tested for antifungal activity; the
method used was cup plate agar diffusion method (Kavanagh,
1972) with minor modifications. Plant extracts were tested against
plant
pathogens
Aspergillus
niger
(ATCC9763-8/29/2005),
Nattrassia mangiferae (Eltahir, 2003), Aspergillus flavus, (N, H, L,
2006). Fusarium moniliforme, (PPL/PPA/MA, 2006).
3.5.1 Preparation of Fungal suspensions
One ml aliquots of a 24 hours broth culture of test fungus were
aseptically distributed into sabouraud dextrose agar slant and
incubated at 25oC for 4 days. The fungal growth was harvested and
washed with sterile normal saline and finally suspended in 100ml
of normal saline and the suspension was stored in the refrigerator
of 4o C until used (Kavanagh, 1972).
3.5.2 Testing for the antifungal activity, cup-plate agar diffusion
method
The total activity of T. brownii crude extract was carried out by
cup-plate agar diffusion method to assess the antifungal activity of
the prepared extracts. 20 ml of aliquots of the inoculated sabouraud
dextrose agar were distributed into sterile Petri dishes. The agar
was left to set and in each of these plates 4 cups (10 mm in
diameter) were cut using borer tool 10 mm in diameter. Alternative
cups were filled with 0.1ml from the different diluted extracts
(petroleum ether, Chloroform, and methanolic extracts) using
automatic micropipette. Five replicates for each extracts of the
tested fungi were made each test. Two different concentrations of
37
the Plant extracts were used: 1mg in 1ml solvent and 5mg in 1ml
solvent. The extracts were allowed to diffuse at room temperature
for two hours. The plates were then incubated at 25º C for 24
hours.
After incubation the diameters of the resultant growth inhibition
zones were measured, average was taken and the mean values were
tabulated. The solvents used for extraction and dissolution were
used as negative controls by adding them to the media instead of
the extracts in another set of experiment to confirm that they have
got no effect on the growth of the fungi. Further investigations
were conducted with concentration on the ethyl acetate extract
only.
3.5.3 The antifungal activity by determining the minimum
inhibitory concentration (MIC)
To quantifying the activity of the extracts, the modified serial
dilution method was used to determine the minimum inhibitory
concentration (MIC) (Abdalla, 2004). The minimum inhibitory
concentration (MIC) of the extracts was estimated for each of the
test organisms in triplicates. Two grams of the extract were
dissolved in 20ml-distilled water to end with the one of 0.1g/ml,
five serially dilution of the one were made to have the following
concentration 0.05g/ml, 0.025g/ml, 0.0125g/ml, 0.0062g/ml and
0.0031g/ml. The 10ml serially diluted extracts were added to 10ml
sabouraud dextrose agar in petridishes. Each dish was then
inoculated with 0.1ml of the spore suspension of A. niger, A.
flavus, N mangiferae and F. moniliforme, Petridishes were
incubated at 250 C and examined for growth after 24h. The least
concentration of the plant extracts that does not permit any visible
38
growth of the inoculated test organism in the broth medium was
regarded as the MIC in each case. Control experiments were
performed using the solvents only without the plant extracts.
39
CHAPTER FOUR
RESULTS AND DISSCUSION
4.1 Thin layer chromatography of the bark and the wood of T.
brownii
All analyzed extracts gave negative reaction with Dragendorff
reagent suggesting the absence of alkaloids. Alkaloid develops
brown or orange visual day light zones immediately on spraying
(Wagner, et.al., 1984). Typical pink to purple colours were
developed upon spraying with vanillin H2SO4 (heat 110˚C) in all
studied extracts (Table 3) Flavonoids were mainly accumulated in
the ethyl acetate fraction of both studied parts (wood and bark).
The selection of a suitable stationary phase and solvents depends
on the class of fractions. Vanillin H2SO4 is a universal reagent that
detects components of essential oils, terpenoids, stilbenes, phenols
etc. (Wagner, et.al., 1984). Metabolites to be examined (Robards,
and Antolovich, 1997). Best separation was obtained using RPTLC (Merck), and flavonoids were detected using natural product
reagent (NPR) (Fig 6. Table 4). The presence of flavonoids was
confirmed by their colour change from quenching fluorescence
(366 nm) to yellow or orange colour and prominent blue color in
case of flavonoidal acids or other phenolic acids (366) after
spraying with natural product reagent (NPR) (Fig 6 Table 4).
Fluorescence behavior of flavonoids in response to (NPR) is
structure dependent. Flavonols e.g. glycosides of quercetin and
myricitin develops orange color and those of kaempferol and
isorhamnetin yellow to green color flavones glycosides of luteolin
40
develops orange colour and those of apigenin yellow to green
(Wagner, et.al., 1984).
41
Table: 3 TLC profile for the ethyl acetate fraction of T. brownii
bark and wood after vanillin H2SO4 Reagent
Spot No.
Rf
UV reaction λmax
Expected
Vanillin
metabolite
value
254nm
366nm
w1in RP
0.571
Quenching
Blue f
Dark spot
Terpenoids
W2in RP
0.557
Quenching
Blue f
Blue
Flavonoids
W3in RP
0.357
Quenching
Brown
Purple
Terpenoids
b1in RP
0.871
Quenching
Yellow
Pink
Terpenoids
b2in RP
0.571
Quenching
Blue f
Quenching
Terpenoids
b3in RP
0.557
Quenching
Blue
Blue
Flavonoids
b4in RP
0.357
Quenching
Brown
Purple
Terpenoids
w1in NP
0.870
Quenching
Blue f
Pink
Flavonoids
w2in NP
0.797
Quenching
Blue f
-
Flavonoids
w3in NP
0.565
Quenching
Yellow
Blue
Flavonoids
w4in NP
0.507
Quenching
Yellow
Purple
Terpenoids
b1in NP
0.797
Quenching
Blue f
-
Flavonoids
b2 in NP
0.565
Quenching
Blue f
Blue
Flavonoids
b3 in NP
0.507
Quenching
Yellow
Purple
Flavonoids
b4 in NP
0.362
Quenching
Yellow
-
Flavonoids
w in NP = wood normal phase
b in NP = bark normal phase
w in RP= wood in reverse Phase
b in RP = bark in reverse phase
42
Table: 4 TLC profile for the ethyl acetate fraction of T. brownii
bark and wood after NPR Reagent
Spot no
Rf
UV Reaction λmax
NPR
Expected
254nm
366nm
(In366nm)
Metabolite
w1 in RP 0.931
-
Blue f
Blue f
Flavonoidacid
w2 in RP 0.857
Quenching
Yellow f
Yellow f
Flavonoid
w3 in RP 0.714
Quenching
Blue f
Blue f
Flavonoid
w4 in RP 0.571
Quenching
Blue f
Blue f
Flavonoid
b1 in RP
0.857
Quenching
Yellow f
Yellow f
Flavonoid
b2 in RP
0.743
Quenching
Blue f
Blue f
Flavonoid
b3 in RP
0.571
Quenching
Blue f
Blue f
Flavonoid
w1in NP
0.857
Quenching
Blue f
Blue f
Flavonoid
w2in NP
0.814
Quenching
Blue f
Blue f
Flavonoid
w3in NP
0.671
Quenching
Blue f
Pink
Flavonoid
w4in NP
0.571
Quenching
Blue f
Blue f
Flavonoid
b1 in NP
0.857
Quenching
Blue f
Blue f
Flavonoid
b2 in NP
0.814
Quenching
Blue f
Blue f
Flavonoid
b3 in NP
0.329
Quenching
Blue f
Pink
Flavonoid
b4 in NP
0.571
Quenching
Blue f
Blue f
Flavonoid
Value
NPR= Natural products reagent
w in NP = wood in normal phase b in NP = bark in normal phase
w in RP = wood in reverse Phase
43
b in RP = bark in reverse phase
A
B
Figure 6-A: TLC Profile in normal phase (NP) Ethyl acetate phases of the studied parts of T. brownii. Sprayed with
NPR (A) 254 and (B) 366nm
44
w1. RP
b1.RP
w2. RP
44
b2.RP
w3.RP
w2. RP
w3. RP
b2. RP
w4. RP
b3. RP
B
A
Figure 6-B: TLC
b3.RP
w4. RP
b1. RP
Profile in Reversed phase (RP) Ethyl acetate phases of the studied parts of T. brownii. (A) 254 nm
and (B) Sprayed with NPR at 366nm
45
4.2 Antimicrobial activity of T. brownii
Cup-plate agar diffusion technique was used to determine the
minimum inhibitory concentration to evaluate the effects of the
extracts on the four standard organisms.
The effects of varying the concentration of the extracts, the type of
the solvent used in the extraction method and the four standard
organisms were the three variables examined for the in vitro
antifungal activity of the wood and the bark of T. brownii extracts.
The wood and the bark extracts of T. brownii at the concentration
of 1mg/ml against the four standard organisms gave inhibitory
zone for the four solvents used except for the PE extract which
exhibited no inhibition zone; the mean diameters were 12mm in
ethyl acetate extract, 13mm in aqueous extract while it was less
than1mm in the case of Ch extract (Table 5).
The following experiments were performed using the plant extract
of the different solvents at the concentration of 5mg/ml:
All the four standard organisms not affected by petroleum ether
extract of the wood and the bark of T. brownii, which indicates that
petroleum ether extract, might not have an antifungal activity.
The average of four readings for the inhibitory zone of Ch extract
of the wood of the plant used was as follows:
12.5mm in A. niger, 14mm in A. flavus, 13mm in both Nattrassia
mangifera, and Fusarium moniliform, but the bark extract of the
same solvent gave inhibitory zone diameter of 11mm against A.
niger, A. flavus and Fusarium moniliform and 10.5mm against
Nattrassia mangifera as shown in table 5.
The aqueous extract of the wood of T. brownii exhibited the
highest antifungal activity against A. niger which 13mm inhibitory
zone diameter, while it was 11mm against A flavus and Natrassia
46
mangifera and 12mm diameter in the case of Fusarium moniliform,
the inhibitory zones of the aqueous extract of the bark were 14mm
against A. niger and A. flavus, and 20mm against Nattrassia
mangifera and Fusarium moniliform (Table 5)
Likewise, the EtoAc extracts of the wood of T. brownii give
growth inhibitory zones with the mean diameter of 15mm against
A. niger, A. flavus, Nattrassia mangifera and Fusarium moniliform,
the EtoAc extract of the bark was found to give antifungal activity
represented by the inhibitory zones of 15mm against A. niger,
Fusarium moniliform and Nattrassia mangiferae, and 14.5mm a
ganist A. flavus (Table 5)
The results of the antifungal effects of the eight extracts of T.
brownii species against the four standard organisms Showed that
the wood extract generally gave more antifungal effects than the
bark extracts (Table 5).
Substantial antifungal effects were found at the concentration of 5
mg/ml compared with 1mg/ml for the different solvents extracts.
The solvents used for extraction and dissolution used as negative
controls instead of the extracts confirm that they have got no effect
on the growth of the fungi.
47
Table: 5 Antimicrobial activity of the wood and bark extracts of T.
brownii
Measurment of inhibitory
Plant
Extract
Parts
Concentration
Wood
Wood
Bark
Bark
1mg/ml
5mg/ml
1mg/ml
5mg/ml
Extracts
zones diameter MIZD for
Fungi (mm)
A.n
A.f
N.m
F.m
Petroleum ether
-
-
-
-
Chloroform
>1
>1
>1
>1
Ethyl acetate
12
12
12
12
Aqueous
13
13
13
13
Petroleum ether
-
-
-
-
Chloroform
12.5
14
13
13
Ethyl acetate
15
15
15
15
Aqueous
13
11
11
12
Petroleum ether
-
-
-
-
Chloroform
>1
>1
>1
>1
Ethyl acetate
12
12
12
12
Aqueous
13
13
13
13
Petroleum ether
-
-
-
-
Chloroform
11
11
10.5
11
Ethyl acetate
15
14.5
15
15
Aqueous
14
14
20
20
A..n = Aspergillus niger. A .f = Aspergillus flavus. N.m = Nattrassia
mangifera and F.m = Fusarium moniliform.
MIZD (mm): >18mm: sensitive
14-18: intermediate
<14mm: Resistant
48
4.3 Minimum inhibitory concentration of T.brownii extracts
(MIC)
Another set of experiments was performed for the evaluation of the
effects of the extracts against the four standard organisms by
determining the minimum inhibitory concentration (MIC).
The modified serial dilution technique was used (Abdalla, 2004).
The ethyl acetate extracts of the bark and the wood of T. brownii at
the concentrations ranging from 0.001g/ml to 0.05g/ml against the
four standard test organisms demonstrated activity against A.
flavus, Nattrassia mangiferae and Fusarium moniliforme, but
against A. niger no effect was obtained even at the higher
concentration used.
The MIC of the ethyl acetate extracts of the wood and the bark of
the plant used against A. flavus, Nattrassia mangiferae and
Fusarium moniliforme could not be determined because even at
concentration as low as, 0.001g/ml antifungal activity was
observed, So the MIC could be considered to be lower than
0.001g/ml. But the extracts against A. niger even at the higher
concentration (0.05g/ml) no effect was shown. This result
demonstrated that the ethyl acetate extracts of the wood and the
bark of T. brownii against A. niger are either not effective or they
may have an MIC above 0.05g/ml.
49
4.4 RP-HPLC-DAD of T. brownii wood ethyl acetate phase
Reverse phase HPLC-DAD of the ethyl acetate phases of the wood
and bark are presented in figure (7). Stilbenes and flavonoids are
mainly accumulated in the ethyl acetate extract of the wood, (Fig.
9). (Fig. 10) Show that the stilbenes are best detected at 320 nm.
The utility of RP HPLC separation for more specific and selective
identification of stilbene and flavonoid derivatives was greatly
enhanced by mass-spectrometric detection; in particular the use of
MS–MS enabled the safe identification of co-eluting peaks in the
complex biological matrix, (Stecher, et.al., 2001).
A
comparison
RP-HPLC
(λmax
254nm
and
320
-370)
chromatograms are presented in (Fig. 7 and 8). This UV range
enabled the detection of the metabolites classes of interest
(flavonoids and stilbenes). It is clear from these chromatograms
that similar compounds exists among the active extracts, namely
the ethyl acetate phases of the wood and bark of the plant studied.
Accordingly, the wood ethyl acetate fraction was subjected to
further analysis for identification of the major compounds with
special emphasis on its flavonoids and stilbenes content. Solid
phase extraction led to a further step in the purification of the wood
ethyl acetate phase.
50
Figure: 7 RP-HPLC-DAD Chromatogram of ethyl acetate fraction of T. brownii wood (A) and bark (B) recorded at
λmax 254nm
51
C
Figure: 8 RP-HPLC-DAD Chromatogram of ethyl acetate fraction of T. brownii bark (A) and wood (B) recorded at
λmax 320-380 nm, (C) Figure 6.B.B
52
Figure 9: RP- HPLC-DAD chromatogram of ethyl acetate fraction of T. brownii wood recorded at λmax 254(A) and
320(B) nm
53
4.4.1 Identification of compounds in T. brownii wood ethyl
acetate phase by LC–triple quadruple mass spectrometric
analysis (LC-MS/MS)
Flavonoids were identified using UV profile at 360 nm and their
mass fragmentation pattern using LC-MS/MS in comparison with
reported data. The most useful fragmentations in terms of
flavonoid aglycone identification are those that require cleavage of
two C-C bonds of the C-ring resulting in structurally informative
i,j
A+ and i,jB+ ions (Fig. 11). These ions can be rationalized by retro-
Diels-Alder (RDA) reactions and are the most diagnostic fragments
for flavonoid identification since they provide information on the
number and type of substituents in the A- and B- rings, Cuyckens
and Claeys (2004). For free aglycones, the i,jA and i,jB labels refer
to the fragments containing intact A- and B-rings, respectively, in
which the superscripts i and j indicate the C-ring bonds that have
been broken. The major routs of fragmentation resulting in A and
B ions require cleavage of the C-C bonds at positions 1/3, 0/2, 0/3,
0/4 or 2/4 of the C-ring (Fig. 10). The fragmentation pathways
depend strongly on the substitution pattern and the class of
flavonoids studied, e.g. the additional hydroxyl group in position 3
of flavonols results in more and different possibilities for
fragmentations compared with flavones.
2H, and
1,3
0,2
A+,
0,2
A+ - CO,
1,4
A+ +
B+ - 2H are typically observed for flavonols, while
1,3
B+, 0,4 B+, and 0,4 B+ - H2O are found in flavones. Product ions from
glycoconjugates are denoted according to the nomenclature
reported by Cuyckens and Claeys (2004). Ions containing the
aglycone are labeled k,lXj, Yj, and Zj, where j is the number of the
interglycosidic bond broken, counting from the aglycone, and the
54
Superscripts K and l indicate the cleavages within the carbohydrate
rings. The glycosidic bond linking the glycan part to the aglycone
is numbered 0.
55
0,2
X1
Y1
0,2
X0
Z1
OH
O
O
B0
Z0
CH2
OH
OH
1,3
Y0
OH
B
O
O
OH
0
O1
2
OH
0,2
A1
C
A
OH
3
4
B1
0,2
A2
OH
O
B2
1,3
A0
Figure: 10 Fragmentation pathways for flavonoid glycosides
(illustrated on apigenin-7-O-rutinoside) (Cuyckens and Claeys,
2004).
55
4.4.2 Compounds structures assignment in the ethyl acetate
phase of the wood of T. brownii
Assignment of structures of the metabolites recorded in the wood
ethyl acetate phase was done by studying the results of LC-MS/MS
CID experiments fragments and comparing them to the reported
data or to injected standards when available (Table 6. Fig. 11)
The overall polarity and stereochemistry of the compound are key
factors governing their chromatographic behaviour. It has been
found that sugars with a D-configuration namely glucose,
galactose, xylose and glucuronic acid are usually linked to the
aglycone by β bonds, whilst α linkages occur to L- arabinose and
L-rhamnose (Cuyckens and Claeys, 2004).
Compound 1,2 (m/z 469,491) are most polar compound (6.8min).
Fragmentation of the ({M-H}- - 44) from the main molecule
suggests the loss of COOH group. According to the fragmentation
pattern of the product ion after the loss of the acidic group and in
comparison with published data (Angelika, et.al., 2002, Van der
Doele 1998) Compound 1 was assigned masilinic acid and
compound 2 asistic acid. Both pentacyclic terpenoid acids were
reported previously from the genus Terminalia (Garcez, et.al.,
2003).
Compound 3 and 4 (m/z 541) showed similarity in fragmentation
pattern with retention time 11.1min and 13.4 min, respectively.
Both compounds possessed of a product ion of m/z 227 ({M-H}after the loss of a 314 fragment attributed to a galloylhexose
molecule. Fragmentation pattern of this product ion {m/z 227} was
in agreement with that of the stilbene resveratrol. Hence, these
compounds were assigned resveratrol 3-O-β-galloylglucoside.
Differences in their retention times suggest them to be isomers of a
56
cis (compound 3) and trans (compound 4) form, (Joseph, et.al.,
2007). Resveratrol was previously isolated from Terminalia
sericeae
(Joseph,
et.al.,
2007)
but
resveratrol
3-O-β-
galloylglucoside is reported for the first time in this genus.
Loss of the fragment of m/z 302 form compound 5 [Rt14.4min
(601m/z)] gave products ion of (Y0=271). Fragmentation pattern of
the products ion was in agreement with that of a flavonone (Lee,
et.al., 2002). The compound was suggested to be Flavellagic acid.
The compound was tentatively assigned the structure (appendix 5).
Compound 6 (433 m/z, Rt 15.3 min) first fragmentation pattern
(433{M-H}- -132) gave the products ion (Y0=271) after the loss of
methyl group 15 units. The product ion fragmentation was again
that of naringenin (Lee, et.al., 2002). Regarding the intensity of the
product
ion
compound
6
was
assigned
naringenin
4'
methoxy7arabinoside.
Compound 7 (625 m/z, 16.5 min) is the major compound in the
ethyl acetate extract of T.brownii wood and bark. Loss of two
glucose molecules ({M-H}- -162-162) gave the main peak of the
product ion a glycone (m/z 301). Intensity of the glycone product
ion suggests compound 7 to be quercetin 7-β-0-diglucoside,
Cuyckens and Claeys (2004).
The MS/MS data compound 8 (633m/z, 18.2min) is presented in
Table 8. Loss of a glucose molecule ({M-H}- -162) in addition to
the fragmentation pattern of the product ion suggest the compound
to be. arjunic glycoside. Arjunic glycoside was previously reported
in Terminalia arjuna (Ghosh, et.al., 2008).
Compound 9 MS/MS data are shown in Table 4 (585 m/z.18.4
min) a glycone product ion peak (301m/z) was obtained after the
loss of a pentose sugar molecule and a galloyl molecule {(M-H}- 57
132-153}. Compound 9 was suggested to be Quercetin derivatives.
Comparing the fragmentation pattern of the aglycone product ion
to reported data (Ram, et.al., 1997) and the intensity of this ion
suggest compound 9 to be Quercetin 7-0 - galloyl glucoside.
Loss of 3-methyl molecule for compound 10 (m/z 343. Rt
25.5min) ({M-H}- -15 x 3) with the intensity of the product ion
suggest it to be 5,6 dihydroxy 3', 4’, 7 trimethoxy flavone.
58
Figure 11: RP-HPLC-DAD Chromatogram of ethyl acetate fraction of T.brownii wood extract at λmax 320-380 nm
59
Table (6) Peak No. (Fig 11), RP- HPLC data (Rt), molecular weight (m/z), MS/MS data (m/z) and assigned structures
of the wood of T. brownii ethyl acetate fraction
Compound
Peak
Rt
(min)
M+H
(m/z)
1
2
3
4
5
6
7
8
9
10
6.8
6.8
11.1
13.4
14.4
15.3
16.5
18.2
18.4
25.5
469
491
541
541
601
433
625
633
585
343
CID Mn
Main fraction ions
(m/z)
425,407,379,353,300,271
447, 429, 410, 301
532,424,300,273,227,199,169
532,424,299,275.227
296,270.7,242.8,214.8,
314,229,271
300,256.7,229,185,129
481,463,421,387,305,211
301,283,256,228,785
327,313,298,285
a glycon underli
60
Expected compound
Masilinic acid
Asistic acid
Resevertrol derivative cis
Resevertrol derivative trans
Flavellagic acid ester
Naringenin 4'methoxy 7arabinoside
Querctin 7- ß -0 -diglycoside
Arjunic glycoside
Quercetin7-0 -galloyl glycoside
5,6 dihydroxy 3',4’,7 trimethoxy flavone
CHAPTER FIVE
CONCLUSIONS AND RECOMMENDATIONS
Combretaceae is known for its medical uses in Africa and Asia. To date no
phytochemical records are available for Terminalia brownii. This study was
conducted for phytochemical analysis of two different parts (wood, bark) of T.
brownii. Plant studied different parts extracts were subjected to biological and
chemical screening implementing different chromatographic analytical
methods (TLC, HPLC and LC-MS/MS). The main objectives of this study
were the chemical and biological screening of the parts of the plant studied.
Regarding their well documented biological activities, flavonoids, stilbenes
and phenantherenes were the targeted metabolites in this study. These
metabolites were mainly traced in the most active extracts of the wood of T.
brownii.
5.1 Conclusions
• The results of the antifungal effects of the extracts of T. brownii
showed that the wood extract generally gave more antifungal
effects than the bark extracts. No significant activity was observed
from the petroleum ether extracts of the bark and wood against four
tested organisms
• Substantial antifungal effects were found at the concentration of 5
mg/ml compared with 1mg/ml for the different solvents extracts.
The ethyl acetate extracts of the different parts tested is the most
active among other extracts.
• The MIC of the ethyl acetate extracts of the wood and the bark of
T. brownii against Aspergilus niger are either not effective or they
may have an MIC above 0.05g/ml.
61
• The MIC of the ethyl acetate extracts of the wood and the bark
could be considered to be lower than 0.001g/ml against A. flavus,
Nattrassia mangiferae and Fusarium moniliforme.
•
RP-HPLC-DAD coupled with tandem mass spectrometry was
used for qualitative and quantitative determination of stilbenes,
flavonoids and phenantherenes in ethyl acetate extracts of wood
and bark of T. brownii
• Flavonoids identified include quercitin and naringenin derivatives
and a methoxylated flavone
• Resveratrol galloylglucoside in a cis and trans form was also
identified in the active extract
• Among the compounds identified were pentacyclic terpenoidal
acids which were previously reported in the genus Terminalia
• Present study will help us in promoting and utilizing eco-friendly
preservative in appropriate quantity for specialized and uses.
5.2 Recommendations
• It is expected that the major area of use will be for prevention
purpose, especially in Implementing wood preservation
• Use of active extracts for biological control in forest products.
• Running similar analysis on the roots, leave, seeds of the plant.
• Conserve the biological and genetic diversity of important natural
resources.
• Genomic mapping of the plant studied.
62
CHAPTER SIX
REFERENCES
Abdalla, A. N. 2004. Antimicrobial and wound healing activity of ten
medicinal plants. MSc, thesis submitted to University of Khartoum
Abdelhameed, Z. 2003. Effect of relative humidity, species and
extractives on the Equilibrium Moisture content of some hard
wood species growing in Sudan. MSc, thesis submitted to
university of Khartoum
Abdurazag. A.; G. Thomas. ; Hartley.; and Peter, G. W. 1997.
Distribution of flavonoid, alkaloids acetophenones and
phologlucinols in the genus Bosisloa(Rutaceae).Journal of
Biochemical systematics and ecology,Vol. 25 (7). Pp 611-617.
Adyana, K.; Y. Tezuka; S. Awale; H. Banskota; Tan, K.Q.; and S.
Kodata. 2000. Six triterpen glycoside from the seeds of
Combretum. Journal of phytochemistry. Vol. 54. Pp 795-799
Ahmad, I.; Zafar, M.; and F. Mohammad. 1998. Screening of some
Indian medicinal plants for their antimicrobial properties. Journal
of Ethnopharmacology, Vol. 62. Pp 183-193.
Angeh, J. E.; X. Huang; G. E. Sattler; H. Swan.; A. Dahase.; and J.N.
Eloff, 2006. Antimicrobial and anti-inflammatory activity of four
known and one new triterpenoid from Combretum imberbe
(combretaceae). Journal of Ethnopharmacology, Vol. 110. Pp5660
Angelika otto.; Bernd. R. T. S.; Volker. W.; Lutz. K.; and Withelon. P.
2002. TerpenoidsCompositionof three fossil resin from Cretaceae
and tertiary Conifers. Renew of palaeobatony and palynology.Vol.
120. Pp203-215.
Arbabi, M.; S. Nasseri; A. R. Mesdaghinia.; S. Rezaie.; K. Naddafi.
G. H. Omrani.; and M. Yunesian. 2004. Survey on physical,
chemical and microbiological characteristics of PAH-contaminated
soils in Iran. Iranian Journal of Environmental Health Science and
Engineering, Vol. 1 (1): Pp 30-37
Baba-Mousa, F.; Akpagana, K.; and P. Bouchet. 1999. Antifungal
activity of seven West African combretaceae used in traditional
medicine. Journal of Ethnopharmacology, Vol. 66. Pp 335-338.
63
Cao, S. Peggy.; J. Brodie.; J. S. Miller.; R. R. Fidy.; R. Chris.; B.
Rabodo.; A. Vincent.; E. Rasamison.; and G. K. David. 2008.
Antiproliferative xanthones of Terminalia calcicola from the
Madagascar rain forest. Journal of Natural Products, Vol 70 (4):
Pp 679–681.
Cassidy, A.; B. Hanley.; and M.R. Lamuela-Raventos. 2000.
Isoflavones, lignans and stilbenes origins, metabolism and potential
importance to human health. Journal of the Science of Food and
Agricultur, Vol 80 Pp 1044-1062
Conrad, J.; B. Vogler.; I. Klaiber.; G. Roos.; U. Walter.; and W.
Kraus. 1998. Two triterpene esters from Terminalia macropetra
bark, Journal of phytochemistry, Vol 48 (4): Pp 647-650
Cown, M. M. 1999. Plant Products as Antimicrobial Agents, Clinical
Microbiology reviews, Vol. 12. Pp 4564-582.
Cousin, D.; J. Mann.; M. Nieuwenhuyzen.; and H. V. D. Berg. 2006.
A new approach to combretastatin D2. Journal Organic and
Biomolecular; Chemistry, Vol. 4, Pp 54-62
Cuyckens, F.; and M, Claeys. 2004. Mass spectrometry in the structural
analysis of flavonoids. Journal of mass spectrometry, Vol. 39 Pp 115
Dahlgren, R.; and R.F. Thorne. 1984. The Order Myrtales:
Circumscription, Variation, and Relationships. Annuals of the
Missouri Botanical Garden, Vol. 71 Pp 633-699.
Devi, R.; S. Narayan.; G. Vani.; and Devi, S . 2007. Astroprotective
effect of Terminalia arjuna bark on diclofenac sodium induced
gastric ulcer. Chemico.biological inters action, Vol. 167 Pp 71-83
Elamin, H. M. 1990. Trees and Shrub of the Sudan, Ithaca Press Exeter.
Pp 91-93.
El Gazali, G. B.; M. S. El Tohami.; A. B. El Egami.; W.S. Abdalla.;
and M.G. Mohammed. 1997. Medicinal Plants of the Sudan,
Medicinal plants of Northern Kordofan.Omdurman Islamic
Universit press, Khartoum, Sudan.IV 7
Eltahir, S. E. 2003, An Investigation on Biological control of Nattrassia
mangiferae using Trichoderma spp in Fiucs spp. MSc. thesis
submitted to University of Khartoum.
64
Gao, H.; Y. Huang.; B. Gao.; Li, P.; C. Inagaki.; and Kawabata, J.
2007. Inhibitory effect on alpha glucosidase by the fruits of
Terminalia chebula Retz. Journal of Bioscience, Biotechnology
and Biochemtry, Vol. 72. Pp 601–60
Garcez, F.; W. Garcez.; D. Miguel.; A, Serea.; and F. Prado. 2003.
Chemical constituents from Terminalia glabrescens. Journal of
Barzil Chemistry Society, Vol. 14 (3): Pp 241-246
Garcez, F.; W. Garcez.; A. Santana.; M. Alves.; M. Matos.; A.
Scaliante. 2006. Bioactive Flavonoids and Triterpenes from
Terminalia fagifolia (Combretaceae). Journal of Brazil Chemistry
Society, Vol.17 (7): Pp 1223-1228
George, N.A. 2004. Plant Pathology. Four editions. Academic press.
Washington. Pp 481-482
Ghosh, B.S and Kadam.U.S. 2008.Antibacterial principles from the bark
of T.arjuna.Current Science, Vol. 94 (1): Pp 27-28
Grova, N.; F. Monteau.; Le. B. Bizec.; C. Feidt.; F. Andre.; and G.
Rychen. 2005. Determinayion of phenantherenes and hydroxy
phenantherenes in various biological matrices at trace levels using
gas chromatography-mass spectroscopy. Journal of analysis
toxicol, Vol. 29 (3): Pp175-81.
Grotewold, W. 2006. The Genetics and Biochemistry of Floral Pigments.
Plant Biotechnology Center, Ohio State University, Columbus,
Vol. 57. Pp 768-772
Harborne, J. B. 1988. Phytochemical Methods. A Guide to Modern
Techniques of Plant Analysis. Second Edition. Routledge,
Chapman and Hall, New York, Pp 288.
Harborne, J. B.; H. Baxter.; and G. P. Moss. 1999. Photochemical
Dictionary: A Handbook of Bioactive Compounds From Plant 2nd
Edition, CRC press.
Hussein, S. G. 2006. A forestation in Arid Lands with Particular Reference
to the Sudan. University of Khartoum Press. Khartoum, Sudan
Jong.; and Gantt. 1987. Aspergillus niger final risk assassment.
Biotechnology program under substances control Act(TSCA) U.S
Enviromental protection agency.
65
Joseph, C. C. ; M.J. Moshi.; E. Innocent.; and M. H. H. Nkunya. 2007.
Isolation of a stilbene glycoside and other constituents of
Terminalia sericeae. African Journal of Traditional,
Complimentary and Alternative Medicines, Vol. 4 (4): Pp 383 –
386
Jossang, A.; M. Seuleiman.; E. Maidoa.; and B. Bado. 1996.
Pentacyclic triterpen from Combretum nigricans. Journal of
Phytochemistry, Vol. 41(2): Pp 594-591
Kandil, F. E.; and M. L. Nassar. 1998. Tannin anti-cancer promoter
from Terminalia arjuna protein. Journal of phytochemistry, Vol.
47(8): Pp1567-1568.
Kavanagh, F. 1972. Analytical Microbiology. Academic press, New
York, London, Vol. 11. Pp11.
Kelemu, S.; Cardona, G.; and G. Segura. 2004. Antimicrobial and
insecticidal protein isolated from seeds of Clitoria ternata, a
tropical forage legume. Plant Physiology and Biochemistry, Vol.
42. Pp 867–873.
Khan, M. R.; Kihara, M.; and A. D. Omoloso. 2002. Antimicrobial
activity of Terminalia complanata and Flacourita zippelii. Journal
of Fitoterapia, Vol. 73. Pp 737-740
Koch, P. 1985. Utilization of Hardwoods Growing in Southern Pine Sites,
volume 1. Department of Agriculture, Forest Service. U.S.
Government printing office, Washington, D.C.20402
Kovacs, A.; Vasas. A.; and J. Hohmann. 2007. Natural phenanthrenes
and their biological activity. Journal of phytochemistry, Vol. 69. Pp
1084-1110
Laszlo, M.; Pour, S.M.; Peter, A.; and O. Robert. 2005. A validated
HPLC method for the quantitative analysis of trans-resveratrol and
trans-piceid in Hungarian wines. Journal of chromatographic
science Vol. 43. Pp 445-449
Lee, M. K.; Bok, S. H.; Jeong, T. S.; Moon. S. S.; Lee. S. E.; Park, Y.
B.; and Choi, M. S. 2002.Supplementation of naringenin and it is
synthetics derivatives altars antioxidant enzyme activites of
erythrocyte and liver high cholestrol.fed rats. Journal of
Bioorganic and Medicinal Chemistry, Vol. 10 (7) Pp 2239-2244.
66
Mann, A.; Banso, A.; and L.C. Clifford. 2008. An antifungal property of
crude plant extracts from Anogeissus leiocarpus and Terminalia
avicennioides Tanzania, Journal of Health Research, Vol. 10 (1).
March, R. E.; E. Lewars. C. Stadey.; X. Sheng.; X. Zhao.; and C.
Meteacalfe. 2005. International journal of Mass Spectroscopy,
Vol. 248. Pp 61-85
Martini, N.; and Eloff, J. N. 1998.The preliminary isolation of several
antibacterial compounds from Combretum erythrophyllum
(Combretaceae). Journal of Ethnopharmacology, Vol. 62. Pp 25563.
Martini, N. D.; D. R. Katerere.; and J. N. Eloff. 2004. Biological
activity of five antibacterial flavonoids from combretum
erythrophyllum (combretaceae). Journal of ethnopharmacology,
Vol. Pp 207-212
Mayer, R. 2004. Five bioflavonoid from Calycopteris floribunda
(combretaceae). Journal of phytochemistry, Vol. 65 Pp 593-601
Maulika, K. S. 2005. Terminalia arjuna. Protects rabbit heart against
ischemic. Reperfusion injury: role of antioxidant enzymes and heat
shock protein, Journal of ethnopharmacology, Vol. 96. Pp.403409.
Mohagheghzade, A.; P. Farid,; M. Shams-Ardakani.; and Y. Ghasemi.
2006. Medicinal smoke. Journal of ethnopharmacology, Vol. 108.
Pp161-184
Ncube, N. S.; A. J. Afolayan.; and A. Okoh. 2007. Assessment
techniques of antimicrobial properties of natural compounds of
plant origin: current methods and future trends African Journal of
Biotechnology, Vol. 7 (12): Pp 1797-1806.
Neuwinger, H.D. 2000. African traditional medicine. A dictionary of
Plant Use and Application. Med. Pharm. Press Stuttgart, Germany.
Ogan, A.U. 1972. The alkaloids in the leaves of Combretum micranthum,
Studies in West African medicinal plants. VII, Journal of Planta
Medica. Pp 21 –210
67
Pettit, G.R.; S.B. Sigh,; M.R. Boyd.; E. Hamel.; .R.K. Pettit.; Schmidt
J.m.; and F. Hogan. 1995. Antiplastic agent isolation and
synthesis of Combretastatin A-4, A-5 and A-6. Journal of
Medicinal chemistry, Vol. 38 Pp 1666-72
Preisigmuller, R.; P. Gnau.; and H. Kindl. 1995. The Inducible 9,10Dihydrophenanthrene Pathway: Characterization and Expression of
Bibenzyl Synthase and S-Adenosylhomocysteine Hydrolase.
Journal of Biochemistry and biophysic, Vol. 317 (1): Pp 201-207.
Ram, A.; P. Laria.; R. Gupta.; K. Pradeep.; and V. Sharma. 1997.
Hypocholesterol effects of Terminalia arjuna a tree bark. Journal
of Ethnopharmacology, Vol 55 (3):Pp165-169
Rajput, K.S.; and K.S. Raob 2004. Death and decay in the trees of
Mango (Mangifera indica L.), Journal of microbiological
research, Vol. 162 (3): Pp 229-234.
Rich, J. S. 1975. Chemical control plant disease: An Exciting future.
Annual review of phytopathology,Vol 13 Pp 257-269
Rijke, E. D.; P.Out.; W. M. Niessen.; F. Ariese.; C. Gooijer.; and U.A.
Brinkman. 2006. Analytical seperation and detection methods for
flavonoids;. Journal of chromatography A, Vo1. 112 Pp 31-63.
Robard, K.; and M. Antolovich. 1997. Analitical chemistry of fruit
bioflavonoids. Journal of Analyst, Vol.122. Pp 16-20
Rogers, C. B.; and L. Verotta. 1996. Chemistry and biological properties
of the African Combretaceae. In: Hostettman K, Chinyanganga F,
Maillard M, Wolfender JL. (Eds), Chemistry, Biological and
Pharmacological properties of African Medicinal Plants.
University of Zimbabwi Publications, Harare, Zimbabwe.
Sener, B. 1999. Biodiversity biomelecular Aspects of Biodiversity and
innovative utilization, Kluwer academic/ plenum publishers. New
York.
Schroder, J.; and G. Schroder. 1990. Stilbene and chalcone synthases:
related enzymes with key functions in plant- specific pathway.
Journal of biosience, Vol. 45. Pp 1-8.
Seigler, D. 1998. Plant Secondary Metabolism. Springer Verlag, Pp146148.
68
Stecher, G.; C. Huck.; M. Popp.; and G. K. Bonn. 2001. Determination
of flavonoids and stilbenes in red wine and related biological
products by HPLC and HPLC–ESI–MS–MS. Journal of Analytical
chemistry, Vol. 371. Pp73–80
Stobiecki, M. 2000. Application of mass spectroscopy for identification
and structural studied of flavonoid glycosides, Phytochemistry,
Vol. 54 Pp 237.
Tyhrquist, P. 2007. Traditional medicinal uses and biological activities of
some plant Extracts of African Combretum Loefl. Terminalia L.
and Pteleopsis Engl. Species (Combretaceae). University of
Helsinki.
Tsoumis, G. 1968. Wood as Raw Material. Pergamon Press, Oxford.
Tang, X.; J. Gao.; Y. Wang.; ; Y. Fan.; L. Xu.; X. Zhao.; Q. Xu.; and
Z. Qian. 2005. Effective protection of Terminalia catappa l. leaves
from damage induced by carbon tetrachloride in liver
mitochondria, Journal of Nutritional biochemistry, Vol. 17. Pp
177-182.
Van der Doela. G. A,;V.berg.; K. J. Boon.; J. Jshibaym.; N. Dela Ric.;
and E. C. Genwit. 1998. Analysis of fresh triterpenoid resin and
aged triterpenoid varnishes by HPLC. APCI.MS/MS. Journal of
chromatographyA, Vol. 809. Pp 21-37
Wansi, J. D.; M. C. Lallemand.; D. D. Chiozem.; F..A. Toze.; L. M.
Mbaze.; S. Naharkhan.; M. C. Iqbal.; F. Tillequin.; J. Wandji.;
and Z.T. Fomum. 2007. α-Glycosidase inhibitory constituents
from stem bark of Terminalia superba (Combretaceae), Journal of
phytochemistry Vol. 68 (15): Pp 2096-2100.
Wagner, H.; Bladt, S.; and Zgaink, E. M. 1984. Plant Drug Analysis a
Thin Layer Chromatography. Springer-Verlge, Berlin Heidelberg
New York.
Yadav, R. N.; and K. Rathore. 2000. A new cardenolide from the
roots of Terminalia arjuna. Journal of Fitoterapia, Vol. 72 (4): Pp
459-461
http://www.scielo.br/ (Accessed 2008-07-15). Bioactive compounds in
Terminalia spp
69
http://www.sciencedirect.com Accessed (2007-04-15). The structure of
imberbic acid, alpha. Hydroxy pentacyclic triterpenoid from
Combretum imberbe
http://www.sciencedirect.com (Accessed 2008-12-01). Antineoplastic
agents 338. The cancer cell growth inhibitory. Constituents of
Terminalia arjuna
http: //www.wikipedia.odjechane (Accessed 2008-12-18). Aspergillus
flavus
http: //www.wikipedia.odjechane (Accessed 2008-12-18). Fusarium
moniliforme
http://www.en.wikipedia.org (Accessed 2008-12-18). Aspergillus niger
70
Appendix
ESI MS/MS spectra of compounds isolated from Terminalia
brownii wood:
1. Masilinic acid
2. Asistic acid
3. cis Resveratrol 3-O- ß- galloyl glucoside
4. trans Resveratrol 3-O- ß- galloyl glucoside
5. Flavellagic acid ester
6. Naringenin 4’ methoxy 7 arabinoside
7. Quercitin 7- ß- O diglucoside
8. Quercitin 7-O- galloyl glucoside
9. Arjunic glycoside
10. 5,6 dihydroxy 3’ 4’ 7 trimethoxy flanvon
71
61
enass salih