evaluation of the major flavonoids from acacia nilotica l
TRANSCRIPT
بسم الله الرحمن الرحيم
Evaluation of the Major Flavonoids from Acacia nilotica L.
Soha Salah Ali Sirag
B.Sc. (Hons.) in Scientific Laboratories (Chemistry), Sudan University
of Science and Technology (2009)
A Dissertation
Submitted to the University of Gezira in Partial Fulfillment of
Requirements for the Award of the
Degree of Master of Science
in
Chemistry
Department of Applied Chemistry and Chemical Technology
Faculty of Engineering and Technology
April, 2014
Evaluation of the Major Flavonoids from Acacia nilotica L.
Soha Salah Ali Sirag
Supervision Committee:
Name Position Signature
Prof.Mohamed Abdel Karim Mohamed Main Supervis …………….
Dr.Mohamed Osman Babiker Co-supervisor ……………..
Date: April, 2014
i
Evaluation of the Major Flavonoids from Acacia nilotica L.
Soha Salah Ali Sirag
Examination Committee:
Name Position Signature
Prof.Mohamed Abdel Karim Mohamed Chair Person ……….
Dr.Kamal Mohamed Saeed External Examiner .............
Dr .Mostafa Ohag Mohamed Internal Examine ………..
Date of Examination: 22/4/ 2014
ii
Dedication
Dedicated to:
My Parents Soul ,,,
My Teachers ,,,
Sisters and Friends ,,,
iii
Acknowledgement
I am gratefull to Allah, Almighty , that my work was brought to reality.
Thanks to my supervisor : Prof. Mohamed Abdel Karim Mohamed for his
continual help encouragement and infinite support.
Thanks to my co-supervisor: Dr. Mohamed Osman Babiker for his
suggestions , advice and support.
Thanks are extended to the University of Gezira for facilities. Also I would
like to thank the Dept. of Chemistry , Sudan University of Science and
Technology for the Laboratory facilities .
iv
Evaluation of the Major Flavonoids from Acacia nilotica L.
Soha Salah Ali Sirag
Abstract
Flavonoids are natural products.They are secondary metabolites which play
an important role in plant physiology where they occur both in the free state
and as glycosides. These compounds are yellow or white plant pigments.
Most of the flavonoids possess significant physiological activity. The aim of
this study is to isolate the major flavonoid from the pods of Acacia nilotica
and then conducting some IR and UV studies on the isolate .The extraction
was made by 95% ethanol at room temperature for 48 hr. In paper
chromatography Whatman number 3 sheets were irrigated with 10% acetic
acid. In this way compound I was isolated . The IR spectrum showed
bending and stretching vibrations in the aromatic region which is consistent
with the structure of the targeted phytochemical. It also demonstrated the
C-O stretching characteristic of the pyran ring in flavonoids. It also showed
the presence of C-H and OH stretchings in the hydrogen region. A carbonyl
stretching was also manifested. The UV shift reagent sodium methoxide
indicated the presence of a 4`-OH function, while the shift reagent
aluminium chloride indicated the presence of a 5-OH function.In conclusion
the acumulated spectral data indicated that the isolated flavonoid is a : 5,4`-
dihydroxyaurone . For elucidation of structure of the isolated compound
further spectral data are needed (1HNMR , 13CNMR and MS).
V
هطيفيه عليدراسات وإجراء في ثمار القرض ةالرئيس اتيدنوالفلاف دراسة
صلاح علي سراج سهي
الدراسة ملخص
أو في شكل وقد تكون على هيئة طليقة أهمية في النبات نواتج طبيعية لها يدات ونالفلاف
. تهدف هذه الدراسة إلي . ومعظم الفلافونيدات لها اثار فسيولوجية واضحةجلايكوسيدات
لإجراء يد الرئيسنالقرض ثم فصل الفلافو يدات الموجوده في ثمار نباتنوستخلاص الفلافإ
ياانو ستخدم الاأمراء والأشعة فوق البنفسجية ( . الدراسات الطيفية عليه )طيف الأشعة تحت الح
فى كروموتوغرافيا .فى درجة حرارة الغرفةساعة 48 لمدة يداتنوستخلاص الفلافإ( في 95%)
. أوضحت يد الرئيس فصل الفلافونفتم الخليكحمض %10والمذيب 3الورق استخدم واتمان رقم
يار التى تميز الحلقة الإد الامتصاصات المميزة لرابطة ووجة تحت الحمراء عالأش دراسات مطيافية
روماتية الرابطة الانائية فى الحلقات الأ متصاصاتالمتجانسة فى الفلافونيد. كذلك أوضحت إ غير
اصا مميزا هيدروجين اليفاتية وامتص –عطت امتصاصا لرابطة كربون يضا أأ ورابطة الكربونيل .
زاحة ماوكسيد فقد أوضح كاشف الإلأشعة فوق البنفسجية فية اما مطيالمجموعة الهيدروكسيل. أ
ف الإزاحة كلوريد الالمنيوم فقد ما كاشأ`4 . الصوديوم وجود مجموعة هيدروكسيل فى الموقع
من ثمار يد المستخلص الفلافون. وفى الخلاصة فان 5وضح وجود مجموعة هيدروكسيل فى الموقع أ
النهائي ووضع تركيب ائي هيدوكسي أورون . لتحديد الثن - 5,`4 نبات القرض هو عباره عن
هيدروجين , طيسي للت مال أطياف الرنين النووي المغنساوظيفية نحتاج الي مزيد من الدراالزمر ال
طيسي للكربون وطيف الكتلة .والرنين النووي المغن
vi
Table of Contents
Page No Contents
I Supervision Committee
ii Examination Committee
iii Dedication
iv Acknowledgement
v Abstract
vi ملخص الدراسة
vii Table of Contents
viii Table of contents
Chapter One
1 1. Introduction
1 1.1 General approach
1 1.2 Classification of flavonoids
2 1.2.1 Phenylbenzopyrans
3 1.2.2 Isoflavonoids
4 1.2.3 Neoflavonoids
5 1.2.4 Minor flavonoids
6 1.3 Synthesis of flavonoids
6 1.3.1 Chalcones,dihydrochalcones and racemic flavonoids
7 1.3.2 Asymmetric epoxidation of chalcones
7 1.3.3 α -and β- hydroxyldihydrochalcones
8 1.3.4 Dihydroflavonols
8 1.3.5 Flavan-3-ols and flavan-3,4-diols
9 1.3.6 Isoflavonoids
11 1.3.6.1 Isoflavans
11 1.3.6.2 Isoflavone epoxides
10 1.3.6.3 Isoflavonones
11 1.3.6.4 Petrocarpan
15 1.4 Isolation and identification of flavonoids
17 1.4.1 Preparation of body fluids
18 1.4.2 Nuclear magnetic resonance
19 1.4.3 Mass spectroscopy
20 1.5 Nutraceutical flavonoids
20 1.6 Flavonoids as antioxidant
21 1.6.1 Reactive nitrogen species and inducible nitric oxide synthesis
vii
22 1.6.2 Protective effects of flavonoids
22 1.7 Flavonoids as anticarcinogenesis
26 1.8 Aim of this work 26 1.9 Acacia nilotica
Chapter Two
29 2- Materials and Methods
29 2.1- Materials
29 2.1.1- Apparatus
29 2.1.2- Collection of plant material
29 2.2- Methods
29 2.2.1- Preparation of test reagents for phytochemical screening
30 2.2.2- Preparation of plant extract for phytochemical screening
31 2.2.3- Test for steroids and/or terpenoids
31 2.2.4- Test for alkaloids
32 2.2.5- Test for flavonoids
33 2.2.6- Test for tannins
33 2.2.7-Test for glycosides
33 2.2.8- Extraction of flavonoids from pods of Acacia nilotica
34 2.2.9- Paper chromatography
34 2.2.10- Spectral data of compound I
35 2.2.10.1- UV shift reagents
Chapter Three
36 3- Results and Discussion
36 3.1- Phytochemical screening
37 3.2- Extraction of flvonoids from plant material
37 3.3- Identification of compound I
46 Conclusion
46 Recommendation
References
viii
1- Introduction
1.1. General approach
Flavonoids are natural products found in fresh vegetables with large
concentration and also in other parts of plants . They were called this name
because they are structurally similar to flavone (1)
O
O
(1)
The study of flavonoid chemistry has emerged, like that of most natural
products from the search for new compounds with useful physiological
properties1.
1.2. Classification of flavonoids
The term “flavonoid” is generally used to describe a broad collection of
natural products that include a C6-C3-C6 carbon framework, or more
specifically phenylbenzopyranfunctionality. Depending on the position of
the linkage of the aromatic ring to the benzopyrano (chromano) moiety, this
group of natural products may be divided into three classes: the flavonoids
(2-phenylbenzopyrans)(2),isoflavonoids (3-benzopyrans) (3) and the
neoflavonoids (4-benzopyrans)(4)1:
O2
3
5
6
7
8
4
2`
3`
4`
5`
6`
O
3
(2) (3)
O
4
(4)
1.2.1-Phenylbenzopyrans (C6-C3-C6 Backbone)
Based on the degree of oxidation and saturation present in the heterocyclic
C-ring,the flavonoids may be divided into the following groups:
flavans(5) , flavonones(6) , flavones(7) , flavonols(8), dihydroflavonols(9)
,flavav-3-ols(10) , flavan-4-ols(11) , flavan-3-4-diols(12).
O*
A C
B
O
O
O
O
(5) (6) (7)
O
O
OH
O
O
OH
*
*
O
OH
*
*
(8) (9) (10)
O
*
OH
*
O
*
OH
* OH*
(11) (12)
1.2.2. Isoflavonoids
The isoflavonoids are a distinctive subclass of the flavonoids. These
compounds possess a 3-phenylchroman skeleton that is biogenetically
derived by 1, 2-aryl migration in a 2-phenylchroman precursor.
Isoflavonoids are subdivided into the following groups.
isoflavans(13) , isoflavones(14) , isoflavonones(15) , isoflav-3-enes(16) ,
isoflavanols(17) , rotenoids(18) , coumestanes(19) , 3-arylcoumarins(20) ,
coumaronochmenes(21) , Coumaronochromones(22) , pterocarpans(23)
O
*
2
4
6
8
2`
O
O
(13) (14)
O
O
*
O
O
OH
**
(15) (16) (17)
OO
C B
A
D
78
9
10
11 12
1
2
3
4
5
6
6a
12a*
*
O O
O
A B
C
D1
2
3
4 5
6a
7
8
910
11
11a
(18) (19)
O O
OO
*
(20) (21)
OO
*
O
*
O O
O
A B
C
D
6a
11a
**
(22) (23)
1.2.3. Neoflavonoids
The neoflavonoids are structurally and biogenetically closely related
to the flavonoids and the isoflavonoids and comprise the 4-arylcoumarins (4-
aryl-2H-1-benzopyran-2-ones) (24), 3, 4-dihydro-4-arylcoumarins (25), and
neoflavones. (26).
O
*
O
O
O O
(24) (25) (26)
1.2.4. Minor Flavonoids
Natural products such as chalcones and aurones also contain a C6-C3-C6
backbone and are considered to be minor flavonoids. These groups of
compounds include the 2`-hydroxychalcones (27), 2`-OH-dihydrochalcones
(28), 2`-OH-retro-chalcone s(29),
Aurones (2-benzylidenecoumaranone) (30), and auronols (31)
OH
O
B A
2`
(27)
OH
O
OH
O
(28) (29)
O
O
A
B
O
O
OH
*
(30) (31)
1.3. Synthesis of flavonoids
1.3.1 Chalcones, dihydrochalcones, and racemic flavonoids
Chalcones and dihydrochalcones are considered to be the primary C6-C3-C6
precursors and constitute important intermediates in the synthesis of
flavonoids.
Chalcones are readily accessible via two well-established routes comprising
a base-catalyzed aldol condensation or acid-mediated aldolization of 2-
hydroxyacetophenones(32) and benzaldehydes(33)2 this is usually the
preferred route towards chalcone(34)formation, since under acidic
conditions cyclization of the ensuing chalcone leads to formation of
corresponding racemic flavones(35)3. Dihydrochalcones(36) are generally
obtained via reduction (H2/Pd) of the preceding chalcones
R
OH
O
RH
O
+R
OH
O
R
Base
(32) (33) (34)
R
OH
O
R
2
3`
Acid
R
OH
O
R
H2/Pd
(34) (36)
R
O
O
R
(35)
1.3.2. Asymmetric Epoxidation of Chalcones:
Asymmetric epoxidation of olefinic bonds plays a crucial role in introducing
chirality in the synthesis of several classes of optically active natural
compounds. Wynberg and Greijdanus first reported the utilization of
quininebenzyl chloride(37)(BQC) and quinidine benzyl chloride (BQdC)
(38)as chiral phase transfercatalysts (PTC)4,5.
N
MeO
N+H
Ph
Cl-OH
H
N
MeO
N+H
Ph
Cl-H
HO
(37) (38)
1.3.3. α- and β-Hydroxyl dihydrochalcones
α – and β –hydroxyldihydrochalcones constitute rare groups of C6-C3-C6
metabolites presumably sharing a close biogenetic relationship with the α –
methyldeoxybenzoins and isoflavonoids6 Wynberg prepared an aromatic
deoxy α –hydroxyldihydrochalcone via catalytichydrogenation of the
corresponding chalcone7. Although several procedures, comprising diverse
reagents, such as benzeneselenolate ion, samarium diiodide, aluminium
amalgam/ultrasound, and metallic lithium in liquid ammonia, have been
used for the regioselective reductivering opening of α,β-epoxyketones to
form the β –hydroxyl ketone8 the most general reagent for these conversions
is tri-butyl tin hydride(TBTH)/azobisisobutyronitrile (AIBN)9.
1.3.4.Dihydroflavonols
Although the Algar-Flynn-Oyamada (AFO) protocol10 and the Wheeler
reaction were mainly used for the synthesis of aurones, it was demonstrated
that these reactions can be adapted for the formation of racemic dihydro
flavonols11 in moderate to good yields.
1.3.5.Flavan-3-ols and flavan-3, 4-diols
Flavan-3-ols, (+)-catechin and (-)-epicatechin, represent the largest
class of naturally occurring C6-C3-C6 monomeric flavonoids. Flavan-3-ols
also have received considerable interest over the last few years because of
their importance as the constituent units of proanthocyanidins12.
Progress in the study of these complex phenolics is often hampered by the
limited availability of naturally occurring flavan-3-ol nucleophiles with 2, 3-
trans, and especially 2, 3-cis, configuration. One of the most common ways
for the synthesis of flavan-3-ols and the closely related flavan-3, 4-diol
analogues involves the reductive transformation of dihydroflavonols.
Reduction of the dihydroflavonol (39) with sodium borohydride in methanol
affords the 2, 3-trans-3, 4-trans-flavan-3, 4-diols, while reduction in an
aprotic solvent like dioxane yielded the C4-epimers exclusivelyi3.
MeO
OMe
O
OH
O
OMe
OMe
OMe
NaBH4
MeO
OMe
O
OH
OMe
OMe
OMe
OH
MeOH
Dioxane
MeO
OMe
O
OH
OMe
OMe
OMe
OH
(39)
(40)
(41)
(+)- [13C]-catechin (40) and (-)-[13C]-epicatechin (41) were isolated in high
yield by the formation of their tartaric acid derivatives14.
1.3.6. Isoflavonoids
Synthetic routes to optically pure pterocarpans, exhibiting the
aromatic oxygenation patterns of naturally occurring isoflavonoids, are
limited by the lack of readily accessible starting materials. These restrictions
and the challenge to form the tetracyclic ring system with stereo-control led
to the development of varioussynthetic approaches. Synthetic endeavors
towards pterocarpan comprise arylation15,16 the reduction and cyclization of
the corresponding 2'-hydroxyisoflavanones17 cycloaddition reactions of 2H-
chromenes with 2-alkoxy-1, 4-benzoquinones iiiii and 1, 3-Michael–Claisen
annulation 18.
1.3.6.1.Isoflavans
Given the fact that the configuration at C-3 would dictate the configuration
at C-2 or C-4 in the 3-phenylchroman framework, a series of isoflavones
were synthesized, which would then afford stereoselective access to other
classes of chiral isoflavonoids19. The protocol involved the stereoselective α-
benzylation of phenyl acetic acid derivatives, subsequent reductiveremoval
of the chiral auxiliary, and cyclization into the isoflavones.
1.3.6.2. Isoflavone epoxides
The first representatives of flavone epoxides were prepared either by
alkaline hydrogen peroxide epoxidation of isoflavones or by an
intermolecular Darzens reaction of α-bromo-O-acyloxyacetophenones.
Dimethyldioxirane (DMDO) is a convenient and effective reagent for the
epoxidation of various substituted isoflavones20. Epoxides were obtained in
high yields by utilizing this versatile oxidizing agent. However,
Attempts to synthesize enantiomeric isoflavone epoxides with DMDO and a
chiral auxiliary demonstrated that the sugar chiral auxiliary did not exercise
enantiofacial selectivity and epoxides were isolated as 1:1 diasteromeric
mixtures.
1.3.6.3. Isoflavonones
By employing a stereo-controlled aldol reaction as the key step, optically
active Isoflavones were synthesized by Vicario in good yields21. This
sequence included an asymmetric aldol reaction between (S,S)-(+)-
pseudoephedrine arylacetamide and formaldehyde to introduce chirality in
the isoflavonone carbon framework at C-3.This was followed by the
introduction of the B-ring as phenol ether under Mitsunobu conditions and
subsequent removal of the chiral auxiliary. Acids were then converted by an
intermolecular Friedel–Crafts acylation, yielding the isoflavonone in good
yields and essentially enantio pure.
R3
R1
R2
NPh
OH
O(i)LDA,THF,-78c
(ii)HCOH,THF,-105c
R3
R1
R2
NPh
OH
O
HO
(42)(43)
O
CO2H
R4
R5
(i)Ph3,DIAD,ArOH
(ii)4M H2SO4/Dioxane
reflux
R2
R1
R3
(i)SOCl2/toluenereflux
(ii)SnCl2/CH2Cl2,rt
R4O
R4
R5
R2
R1
R3
R4
AC
BO
(44)(45)
1.3.6.4. Pterocarpan
Despite the identification of the first 6a-hydroxypterocarpan, (+)-pisatin, in
196022, synthetic protocols to these potent phytoalexins are limited by
lengthy multistep routes and a lack of diversity as far as phenolic
hydroxylation patterns are concerned. These confinements are so restrictive
that only two 6a-hydroxypterocarpans, i.e., pisatin and variabilin, have been
synthesized23 the results reported for the stereo selective aldol condensation
between methyl Ketones and aldehydes employing diisopropylethylamine
and chiral boron triflates2iv,25 prompted the investigation for a more direct
synthetic approach to address for the issue of stereo- control at C-6a and C-
11a of the pterocarpan framework and lability and/or stability of protecting
groups under certain reaction conditions. This protocol included
methoxymethyl protection of the benzaldehydes and phenyl acetates as t-
butyl dimethylsilyl (TBDMS) ethers (stable under acidic conditions).
.
R1
R2
OBn
O
TIN/HCLO4
MeOH/rf
(i)H2/Pt/acetone/
rf
(ii)TBDMSCl/imidazol/DMF/rf
OMe
R1
R2
OBn R1
R2
OMe
O O
OTBDMS
(46) (47)(48)
The subsequent condensation between the ester enolates and the benz
aldehydes afforded the 2, 3-diaryl-3-hydroxypropanoates in moderate to
good yields. Cleavage of the silyl ethers using tetrabutylammonium fluoride
(TBAF) on silica26 gave 4-benzylsulfanyl-2`-hydroxyisoflavans, which were
converted to the 6a, 11a-cis-pterocarpans in yields of 39–82% using the
thiophilic Lewis acids,dimethyl (methylthio) sulfonium tetra-fluoroborate
(DMTSF)or silver trifluoromethanesulfonate (CF3SO3Ag)27.
R1
R2
O
OTBDMS
LDA/Et2O
-78c-0c+
R3
H
OMOM
O
R3
OH
MOMO
OOMe
OTBDMS
R2
R1
OMe
R3
OH SBN
OOTBDMS
R2
R1
BnSH/SnCl4/
CH2Cl2/0c
LiAlH4/Et2O
rf
R3
SBN
OTBDMS
R2
R1
OHOHOMe
R3O
SBN
R1
R2
OTBDMS
PPh3/DEAD
rf
R2O
R1
OTBDMS
SBN
TBAF(silica)THF
rf
R2O
R1
OH
SBN
R2O
O
R1
1
2
4
6
7
8
10
11
11a
6a
AgOTf or DMTSF
CH2Cl2/ 0c
1.4. Isolation and identification of flavonoids
Flavonoids and their conjugates form a very large group of natural products.
They are found in many plant tissues, where they are present inside the cells
or on the surfaces of different plant organs.
Flavonoid glycosides are frequently acylated with aliphatic or aromatic
acids. These derivatives are thermally labile and their isolation and further
purification without partial degradation is difficult.
In the plant kingdom, different plant families have characteristic patterns of
flavonoids and their conjugates. All these compounds play important
biochemical and physiological roles in the various cell types or organs (seed,
root,green part, and fruit) where they accumulate28,29,30. Different classes of
flavonoids and their conjugates have numerous functions during the
interactions of plant with the environment.
The identification and structural characterization of flavonoids and their
conjugates isolated from plant material, as single compounds or as part of
mixtures of structurally similar natural products, create some problems due
to the presence of isomeric forms of flavonoid aglycones and their patterns
of glycosylation. A number of analytical methods are used for the
characterization of flavonoids. In many cases nuclear magnetic resonance
(NMR) analyses (H1 and C13) are necessary for the unambiguous
identification of unknown compounds; other instrumental methods(mass
spectrometry, UV and IR spectrophotometry) applied for the identification
of organic compounds fail to provide the information necessary to answer
all the structural questions.
The utilization of various strategies is dependent on the origin of the
biological material from which the target natural products are to be extracted
(plant or animal tissue or body fluids)31.
The utilization of dried plant material for extraction may cause a substantial
decrease in the yield of flavonoid conjugates. Acylated flavonoid glycosides
are especially labile at elevated temperatures and are frequently thermally
degraded during the process of drying plant tissues.
Free flavonoid aglycones exuded by plant tissues (leaf or root) may be
washed from the surface with nonpolar solvents, such as methylene chloride,
ethyl ether, orethyl acetate. However, more polar glycosidic conjugates
dissolve in polar solvents (methanol and ethanol), and these organic solvents
are applied for extraction procedures in Soxhlet apparatus. Mixtures of
alcohol and water in different ratios are applied for the extraction of
flavonoids and their conjugates from solid biological material (plant or
animal tissues and different food products).
The extraction efficiency may be enhanced by the application of
ultrasonication32 or pressurized liquid extraction (PLE), a procedure
performed at elevated temperature ranging from 60oC to 200oC33. However,
the temperature conditions during the extraction procedures have to be
carefully adjusted because of the possibility of thermal degradation of the
flavonoid derivatives.
The choice of the extraction procedure for obtaining flavonoid conjugates
from biological material is very important and depends on the goals of the
conducted research. The evaluation of the spatial distribution of target
compounds on the organ, tissue, cellular, or even subcellular level is of
special interest in some projects.
Multistep chromatographic methods are necessary for the isolation of
individual components from plant extracts containing new uncharacterized
compounds. Various stationary phases are used in column chromatography
including polyamide, sephadex LH-20, and different types of silica gels.
1.4.1 Preparation of body fluids
For the isolation of flavonoids and their derivatives from liquid samples like
beverages (fruit juice) and physiological fluids (blood or urine), two
different approaches are usually applied. The first one is based on liquid–
liquid extraction and the second one on solid-phase extraction of target
natural products mainly on RP C-18 silica gel cartridges.
All physicochemical methods applied in the field of organic chemistry are
useful for structural characterization or identification of individual
flavonoids and their conjugates. The separation approaches mentioned above
may be considered in different ways. The first one is directed toward the
analysis of single compounds obtained after exhaustive isolation and
purification procedures. The method of choice in this approach is H1 and C13
NMR this technique depends on the intensity of the interactions between
different atoms within a molecule placed in a high-intensity magnetic field.
Different NMR experiments have been developed to achieve information
concerning chemical structure of the studied molecule on this basis.
Particularly useful are methods enabling recording of two-dimensional
spectra showing homonuclear interactions [correlation spectroscopy (COSY)
and nuclear overhauser effect spectroscopy (NOESY)] as well as
heteronuclear [heteronuclear single quantum correlation (HSQC) and
heteronuclear multiple bond correlation (HMBC)] to facilitate the
acquisition of all the structural information about an aglycones and the
corresponding sugar substitution.
The NMR spectrometers may be connected on line to liquid chromatographs
(LC-NMR), giving a powerful tool to study mixtures of natural compounds
present in complex samples.
The variety of MS techniques being available in laboratories is a reason that
this technique has a wide range of scientific or practical applications in
biological and medical disciplines.
Important structural data also can be obtained from mass spectra registered
on different types of mass spectrometers (MS). The application of ultraviolet
and infrared spectrophotometers may give valuable information about
specific compounds.
MS applied for the analysis of organic compounds utilize different
ionization methods and may be equipped with different types of analyzers.
In addition, these instruments may be combined with GC/LC or capillary
electrophoresis (CE) apparatus.
1.4.2.. Nuclear magnetic resonance
NMR is a well-established and the most commonly used method for natural
product structure analysis. The studies of flavonoid structures using H1-
NMR were initiated in 1960s34 and along with C13-NMR have become the
method of choice for the structure elucidation of these compounds.
The chemical shifts and multiplicity of signals corresponding to particular
atoms and their coupling with other atoms within the molecule allow for
easy identification of the aglycones structure, the pattern of glycosylation,
and the identity of the sugar moieties present. The literature of this topic is
abundant and rapidly growing35.
1.4.3. Mass spectrometry
Mass spectrometry is a very sensitive analytical method used to
identify flavonoid conjugates or to perform partial structural characterization
using microgram amounts of sample36. Significant structural data can be
obtained from less than 1 mg of the analyzed compound when different MS
techniques are used in combination with chemical derivatization of the
characterized compounds37,75 .
The ionization methods may be divided into two groups differing with
respect to the amount of energy transferred to the molecule during the
ionization process. Electron ionization (EI) belongs to the first group. The
transfer of energy occurs during the interaction of electrons with the
molecule in the vapor state; it may cause the cleavage of chemical bonds and
fragmentation of the molecule, which is characteristic for the analyzed
compound. Other ionization methods deliver lower energy to the studied
molecules during the protonation (positive ion mode) or DE protonation
(negative ion mode) processes. In both cases, the absorbed energy is too low
to cause intense fragmentation. In this situation, techniques of collision-
induced dissociation with tandem MS (CIDMS/MS) have to be applied for
the structural characterization of compounds .The fragmentation
mechanisms are different during high- and low energy collisions used in
electromagnetic or quadruple and ion trap analyzers.
1.5. Nutraceutical flavonoids
“Nutraceutical” is a term coined in 1979 by Stephen DeFelice38. It is
defined “as a food or parts of food that provide medical or health benefits,
including the prevention and treatment of disease.” Subsequently, several
other terms (medical food, functional food, and nutritional supplements)
were used. A nutraceutical is any nontoxic food extract supplement that has
scientifically proven health benefits forboth the treatment and prevention of
disease39 nutraceutical may range from isolated nutrients, dietary
supplements, and diets to genetically engineered “designer” food, herbal
products, and processed products,such as cereals, soups, and beverages. The
increasing interest in nutraceutical reflects the fact that consumers hear
about epidemiological studies indicating that a specific diet or component of
the diet is associated with a lower risk for a certain disease.The major active
nutraceutical ingredients in plants are flavonoids.
The flavonoids are a group of organic molecules ubiquitously distributed in
vascular plants. Approximately 2000 individual members of the flavonoids
group of compounds have been described. As is typical for phenolic
compounds, they can act as potent antioxidants and metal chelators. They
also appear to be effective in cancer. Overall, several of these flavonoids
appear to be effective anticancer promoters and cancer chemo-preventive
agents.
1.6. Flavonoids as antioxidents
Diets high in flavonoids, fruits, and vegetables are protective against a
variety of diseases, particularly cardiovascular disease and some types of
cancer40 Antioxidants and dietary fiber are believed to be the principal
nutrients responsible for these protective effects. Reactive oxygen species
(ROS) are formed in vivo during normal aerobic metabolism and can cause
damage to DNA, proteins, and lipids, despite the natural antioxidant defense
system of all organisms41 ROS contribute to cellular aging42 mutagenesis43
carcinogenesis44 and coronary heart disease45 possibly through the
destabilization of membranes43 DNA damage, and oxidation of low-density
lipoprotein (LDL). Many in vitro studies have demonstrated the potent
peroxyl radical scavenging abilities of flavonoids, which contribute to
inhibiting lipid peroxidation and oxidation of LDL46. Since oxidation of
LDL is implicated in the pathogenesis of coronary heart diseases47 through
its ability to decrease the susceptibility of LDL to oxidation, a number of
researches have undertaken investigations examining the activity of dietary
agents rich in flavonoids in inhibiting LDL oxidation48.
1.6.1. Reactive nitrogen species and inducible nitric oxide synthase
Reactive nitrogen species (RNS) also appear to contribute to the pathology
of cardiovascular diseases. Nitrogen oxide is one RNS- produced by the
action of nitric oxidesynthase in endothelial cells, neurons, and other cell
types. At the sites of inflammation, inducible nitric oxide synthase (iNOS) is
also augmented, and nitric oxide synthesis is further activated. Peroxynitrite,
a potent oxidant generated by the reaction of nitric oxide (NO) with
superoxide in the vascular endothelium, induces LDL oxidation49 and
proinflammatory cytokine-mediated myocardial dysfunction50. Another
potential source of RNS is derived from dietary nitrite,which reacts with the
acidic gastric juice to produce nitrous acid, which decomposes to oxides of
nitrogen. Nitrous acid and its products are able to nitrosate
amines,deaminate DNA bases, and nitrate aromatic compounds including
tyrosine. Several flavonoids and phenolic compounds, including the
epicatechin/gallate family of flavonol, are powerful inhibitors of nitrous
acid-dependent nitration and DNA deamination in vitro51.
1.6.2. Protective effects of flavonoids
The protective effects of flavonoids in biological systems are ascribed to
their capacity to transfer free radical electrons, chelate metal catalysts52
activate antioxidant enzymes53 reduce alpha-tocopherolradicals and inhibit
oxidases54. Green tea is a rich source of flavonoids, primarily catechins and
flavonol. In black tea, as aconsequence of the fermentation process,catechins
are converted to complex condensation products.the. Oral feeding of green
tea leaves to rats resulted in enhanced SOD activity in serum and catalase
activity in liver and an increased concentration of glutathione in the liver55.
Also it was found that theaflavins inhibit xanthine oxidase (XO) and act as
scavengers of super oxides. Theaflavins 3, 3`-digallate (TF-3) inhibited the
superoxide production in HL-60 cells. Therefore, the anti oxidative activity
of tea polyphenols may be due not only to their ability to scavenge super
oxides, but also because of their ability to block XO and relative oxidative
signal transducers56. Other flavonoids such as quercetin,kaempferol,
myristin, apigenin, and leuteolin also have anti oxidative activity in many in
vitro studies57.
1.7- Flavonoids as anticarcinogenesis
Studies on cancer prevention have assessed the impact of a wide variety of
flavonoids and a selected few isoflavones for their efficacy in inhibiting
cancer in a number of animal models. These studies demonstrated that
flavonoids inhibit carcinogenesis in vitro and substantial evidence indicates
that they also do so in vivo58. Flavonoids may inhibit carcinogenesis by
affecting the molecular events in the initiation, promotion, and progression
stages. Animal studies and investigations using different cellular models
suggested that certain flavonoids could inhibit tumor initiation as well as
tumor progression59.
Dietary quercetin inhibited DMBA-induced carcinogenesis in hamster
buccal pouch60 and in rat mammary gland61 when given during the initiation
stage. Quercetin also inhibited DEN- induced lung tumor genesis in mice62
In a medium-term multiorgancarcinogenesis model in rats, quercetin (1% in
the diet) inhibited tumor promotion in the small intestine Feeding rats with
quercetin during either the initiation or promotion stage, inhibited 4-NQO-
induced carcinoma formation in the tongue. Siess and co-workers
investigated the effects of feeding rats with flavone, flavone, tangeretin, and
quercetin on two steps of aflatoxin B1 (AFB1)-induced
hepatocarcinogenesis (initiation and promotion) and found that flavones, and
tangeretin administered through the initiation period decreased cancer risk.
Quercetin decreased oxidative stress-induced neuronal cell membrane
damage more than vitamin C. These results suggest that quercetin, in
addition to many other biological benefits, contributes significantly to the
protective effects of neuronal cells from oxidative stress-induced
neurotoxicity,such as Alzheimer’s disease63. On the other hand, the
suppressive effects of flavones, such as chrysin and a pigenin, on the
expression of the high affinity IgE receptor which plays a central role in the
IgE-mediated allergic response64 has been demonstrate.
Genistein and daidzein (isoflavones derived from soybeans) have been
shown to inhibit the development of both hormone- and non-hormone-
related cancers,including mouse models of breast, prostate, and skin cancer.
Treatment of mice with 100–500 mg genistein/kg diet reduced the incidence
of advanced-stageprostate tumors, in a dose-dependent manner65. A high
isoflavone diet also was shown to inhibit methylnitrosourea-induced prostate
tumor in Wistar rats66. Topically applied genistein reduces the incidence and
multiplicity of skin tumors in the DMBA-initiated and TPA-promoted
multiplicity of skin mouse model by 20% and 50%, respectively66. In the
UVB light-induced complete carcinogenesis model, topical pretreatment of
SKH-1 mice with genistein significantly reduced the formation of H2O2 and
8-hydroxy-2’-deoxyguanosine66.
The anti carcinogenesis effects of green tea, and black tea extracts on
various organs and animal model have been reported67. Studies showed that
green tea polyphenols have a potent inhibitory effect on skin tumorigenicity
in Sensor mice67. A mixture of theaflavin-3-gallate, theaflavin-3′-gallate, and
theaflavin-3, 3`-digallate) reduced NNK-induced lung tumor multiplicity
and volume in A/J mice. These findings are interesting, given the extremely
poor bioavailability of theaflavins, and may suggest that the theaflavins are
metabolized to a more bioavailable active metabolit
In conclusion intensive epidemiological studies have shown consistently that
regular consumption of fruits and vegetables is associated with reduced risk
of chronic diseases such as cancer and cardiovascular disease68. However,
the individual antioxidants of these foods studied in clinical trials, including
β-carotene,vitamin C, and vitamin E, do not appear to have consistent
preventive effects comparable to the observed health benefits of diets rich in
fruits and vegetables69. It has been reported that fresh apples have potent
antioxidant activity; and whole apple extracts inhibit the growth of colon and
liver cancer cells in vitro in a dose dependent manner suggesting that
natural phytochemicals in fresh fruits could be more effective than a dietary
supplement. Apples are commonly consumed and are the major contributors
of phytochemicals in human diets. Apple extracts exhibit strong antioxidant
and ant proliferative activities and the major part of total antioxidant activity
is from the combination of phytochemicals.
Phytochemicals, including phenolic and flavonoids, are likely to be the
bioactive compounds contributing to the benefits of apple. Recent studies
have demonstrated that whole apple extracts prevent mammary cancer in rat
models in a dose-dependent manner at doses comparable to human
consumption of one, three,and six apples per day70.Thus,consumption of
apples may be an effective strategy for cancer chemoprevention.
Chemo-preventive studies have demonstrated that the mechanisms of action
of phytochemicals and nutraceutical in the prevention of cancer go beyond
the antioxidant activity scavenging of free radicals; regulation of gene
expression in cell proliferation, oncogenes, and tumor suppressor genes;
induction of cell cycle asrrestand apoptosis; modulation of enzyme activity
in detoxification, oxidation, and reduction; stimulation of the immune
system; and regulation of hormone metabolism. It is a general theme that the
additive and synergistic effects of phytochemicals and nutraceutical in fruits
and vegetables are responsive for their potent antioxidant and anticancer
activities and that the benefit of a diet rich in fruits and vegetables is
attributed to the complex mixture of phytochemicals and nutraceutical
present in whole foods71.
Recent development in the molecular mechanisms of signal transduction
pathway in various cell systems has provided a strong basis for performing
thesynergistic effects of phytochemicals and nutraceutical in whole foods
that have been ingested by the host. Along this aspect the cancerchemo-
prevention and anti obesity effects of tea and tea polyphenols might
accomplish this through blocking the signal transduction pathways in the
target cells72,73.
1.8-Aim of this work
This work was aimed:
- To screen Acacia nilotica for different phytochemicals.
- To extract the flavonoids from pods of Acacia nilotica.
- To isolate the major flavonoid from the pods of Acacia nilotica.
- To conduct spectral studies (IR and UV) on the isolated component.
1.9-Acacia nilotica
Kingdom: Plantae
Family: Fabaceae
Genus: Vachellia or Acacia
Species: Acacia nilotica
Acacia nilotica tree
Acacia nilotica – flowers
Acacia nilotica –seeds; pods
Acacia nilotica – leaves ,flowers, seed pod
Acacia nilotica commonly known as Egyptian Acacia , is a thorny tree up to
15m in height that produces pods with a characteristic beaded necklace
appearance. The name nilotica originates from its continuous presence in
the Nile vallies where it had been used for tanning and dyeing , as a source
of water – and insect- resistant wood, as a fodder for livestock and in folk
medicine. Different parts of the tree have been used as antiameobic ,
antispasmodic, antidiarrheal and hypotensive. The pods in particular are
used for treatment for fevers, diarrhea, diabetes and skin diseases74.
Phytochemical studies of the aerial parts of the plant resulted in the
identification of a variety phenolic constituents , among which catechin
derivatives were identified. The compounds have a wide range of biological
activities , in particular antioxidant, anticarcinogenic and anti-inflammatory
activities.
1- Introduction
1.1. General approach
Flavonoids are natural products found in fresh vegetables with large
concentration and also in other parts of plants . They were called this name
because they are structurally similar to flavone (1)
O
O
(1)
The study of flavonoid chemistry has emerged, like that of most natural
products from the search for new compounds with useful physiological
properties1.
1.2. Classification of flavonoids
The term “flavonoid” is generally used to describe a broad collection of
natural products that include a C6-C3-C6 carbon framework, or more
specifically phenylbenzopyranfunctionality. Depending on the position of
the linkage of the aromatic ring to the benzopyrano (chromano) moiety, this
group of natural products may be divided into three classes: the flavonoids
(2-phenylbenzopyrans)(2),isoflavonoids (3-benzopyrans) (3) and the
neoflavonoids (4-benzopyrans)(4)1:
O2
3
5
6
7
8
4
2`
3`
4`
5`
6`
O
3
(2) (3)
O
4
(4)
1.2.1-Phenylbenzopyrans (C6-C3-C6 Backbone)
Based on the degree of oxidation and saturation present in the heterocyclic
C-ring,the flavonoids may be divided into the following groups:
flavans(5) , flavonones(6) , flavones(7) , flavonols(8), dihydroflavonols(9)
,flavav-3-ols(10) , flavan-4-ols(11) , flavan-3-4-diols(12).
O*
A C
B
O
O
O
O
(5) (6) (7)
O
O
OH
O
O
OH
*
*
O
OH
*
*
(8) (9) (10)
O
*
OH
*
O
*
OH
* OH*
(11) (12)
1.2.2. Isoflavonoids
The isoflavonoids are a distinctive subclass of the flavonoids. These
compounds possess a 3-phenylchroman skeleton that is biogenetically
derived by 1, 2-aryl migration in a 2-phenylchroman precursor.
Isoflavonoids are subdivided into the following groups.
isoflavans(13) , isoflavones(14) , isoflavonones(15) , isoflav-3-enes(16) ,
isoflavanols(17) , rotenoids(18) , coumestanes(19) , 3-arylcoumarins(20) ,
coumaronochmenes(21) , Coumaronochromones(22) , pterocarpans(23)
O
*
2
4
6
8
2`
O
O
(13) (14)
O
O
*
O
O
OH
**
(15) (16) (17)
OO
C B
A
D
78
9
10
11 12
1
2
3
4
5
6
6a
12a*
*
O O
O
A B
C
D1
2
3
4 5
6a
7
8
910
11
11a
(18) (19)
O O
OO
*
(20) (21)
OO
*
O
*
O O
O
A B
C
D
6a
11a
**
(22) (23)
1.2.3. Neoflavonoids
The neoflavonoids are structurally and biogenetically closely related
to the flavonoids and the isoflavonoids and comprise the 4-arylcoumarins (4-
aryl-2H-1-benzopyran-2-ones) (24), 3, 4-dihydro-4-arylcoumarins (25), and
neoflavones. (26).
O
*
O
O
O O
(24) (25) (26)
1.2.4. Minor Flavonoids
Natural products such as chalcones and aurones also contain a C6-C3-C6
backbone and are considered to be minor flavonoids. These groups of
compounds include the 2`-hydroxychalcones (27), 2`-OH-dihydrochalcones
(28), 2`-OH-retro-chalcone s(29),
Aurones (2-benzylidenecoumaranone) (30), and auronols (31)
OH
O
B A
2`
(27)
OH
O
OH
O
(28) (29)
O
O
A
B
O
O
OH
*
(30) (31)
1.3. Synthesis of flavonoids
1.3.1 Chalcones, dihydrochalcones, and racemic flavonoids
Chalcones and dihydrochalcones are considered to be the primary C6-C3-C6
precursors and constitute important intermediates in the synthesis of
flavonoids.
Chalcones are readily accessible via two well-established routes comprising
a base-catalyzed aldol condensation or acid-mediated aldolization of 2-
hydroxyacetophenones(32) and benzaldehydes(33)2 this is usually the
preferred route towards chalcone(34)formation, since under acidic
conditions cyclization of the ensuing chalcone leads to formation of
corresponding racemic flavones(35)3. Dihydrochalcones(36) are generally
obtained via reduction (H2/Pd) of the preceding chalcones
R
OH
O
RH
O
+R
OH
O
R
Base
(32) (33) (34)
R
OH
O
R
2
3`
Acid
R
OH
O
R
H2/Pd
(34) (36)
R
O
O
R
(35)
1.3.2. Asymmetric Epoxidation of Chalcones:
Asymmetric epoxidation of olefinic bonds plays a crucial role in introducing
chirality in the synthesis of several classes of optically active natural
compounds. Wynberg and Greijdanus first reported the utilization of
quininebenzyl chloride(37)(BQC) and quinidine benzyl chloride (BQdC)
(38)as chiral phase transfercatalysts (PTC)4,5.
N
MeO
N+H
Ph
Cl-OH
H
N
MeO
N+H
Ph
Cl-H
HO
(37) (38)
1.3.3. α- and β-Hydroxyl dihydrochalcones
α – and β –hydroxyldihydrochalcones constitute rare groups of C6-C3-C6
metabolites presumably sharing a close biogenetic relationship with the α –
methyldeoxybenzoins and isoflavonoids6 Wynberg prepared an aromatic
deoxy α –hydroxyldihydrochalcone via catalytichydrogenation of the
corresponding chalcone7. Although several procedures, comprising diverse
reagents, such as benzeneselenolate ion, samarium diiodide, aluminium
amalgam/ultrasound, and metallic lithium in liquid ammonia, have been
used for the regioselective reductivering opening of α,β-epoxyketones to
form the β –hydroxyl ketone8 the most general reagent for these conversions
is tri-butyl tin hydride(TBTH)/azobisisobutyronitrile (AIBN)9.
1.3.4.Dihydroflavonols
Although the Algar-Flynn-Oyamada (AFO) protocol10 and the Wheeler
reaction were mainly used for the synthesis of aurones, it was demonstrated
that these reactions can be adapted for the formation of racemic dihydro
flavonols11 in moderate to good yields.
1.3.5.Flavan-3-ols and flavan-3, 4-diols
Flavan-3-ols, (+)-catechin and (-)-epicatechin, represent the largest
class of naturally occurring C6-C3-C6 monomeric flavonoids. Flavan-3-ols
also have received considerable interest over the last few years because of
their importance as the constituent units of proanthocyanidins12.
Progress in the study of these complex phenolics is often hampered by the
limited availability of naturally occurring flavan-3-ol nucleophiles with 2, 3-
trans, and especially 2, 3-cis, configuration. One of the most common ways
for the synthesis of flavan-3-ols and the closely related flavan-3, 4-diol
analogues involves the reductive transformation of dihydroflavonols.
Reduction of the dihydroflavonol (39) with sodium borohydride in methanol
affords the 2, 3-trans-3, 4-trans-flavan-3, 4-diols, while reduction in an
aprotic solvent like dioxane yielded the C4-epimers exclusivelyiv3.
MeO
OMe
O
OH
O
OMe
OMe
OMe
NaBH4
MeO
OMe
O
OH
OMe
OMe
OMe
OH
MeOH
Dioxane
MeO
OMe
O
OH
OMe
OMe
OMe
OH
(39)
(40)
(41)
(+)- [13C]-catechin (40) and (-)-[13C]-epicatechin (41) were isolated in high
yield by the formation of their tartaric acid derivatives14.
1.3.6. Isoflavonoids
Synthetic routes to optically pure pterocarpans, exhibiting the
aromatic oxygenation patterns of naturally occurring isoflavonoids, are
limited by the lack of readily accessible starting materials. These restrictions
and the challenge to form the tetracyclic ring system with stereo-control led
to the development of varioussynthetic approaches. Synthetic endeavors
towards pterocarpan comprise arylation15,16 the reduction and cyclization of
the corresponding 2'-hydroxyisoflavanones17 cycloaddition reactions of 2H-
chromenes with 2-alkoxy-1, 4-benzoquinones iviv and 1, 3-Michael–Claisen
annulation 18.
1.3.6.1.Isoflavans
Given the fact that the configuration at C-3 would dictate the configuration
at C-2 or C-4 in the 3-phenylchroman framework, a series of isoflavones
were synthesized, which would then afford stereoselective access to other
classes of chiral isoflavonoids19. The protocol involved the stereoselective α-
benzylation of phenyl acetic acid derivatives, subsequent reductiveremoval
of the chiral auxiliary, and cyclization into the isoflavones.
1.3.6.2. Isoflavone epoxides
The first representatives of flavone epoxides were prepared either by
alkaline hydrogen peroxide epoxidation of isoflavones or by an
intermolecular Darzens reaction of α-bromo-O-acyloxyacetophenones.
Dimethyldioxirane (DMDO) is a convenient and effective reagent for the
epoxidation of various substituted isoflavones20. Epoxides were obtained in
high yields by utilizing this versatile oxidizing agent. However,
Attempts to synthesize enantiomeric isoflavone epoxides with DMDO and a
chiral auxiliary demonstrated that the sugar chiral auxiliary did not exercise
enantiofacial selectivity and epoxides were isolated as 1:1 diasteromeric
mixtures.
1.3.6.3. Isoflavonones
By employing a stereo-controlled aldol reaction as the key step, optically
active Isoflavones were synthesized by Vicario in good yields21. This
sequence included an asymmetric aldol reaction between (S,S)-(+)-
pseudoephedrine arylacetamide and formaldehyde to introduce chirality in
the isoflavonone carbon framework at C-3.This was followed by the
introduction of the B-ring as phenol ether under Mitsunobu conditions and
subsequent removal of the chiral auxiliary. Acids were then converted by an
intermolecular Friedel–Crafts acylation, yielding the isoflavonone in good
yields and essentially enantio pure.
R3
R1
R2
NPh
OH
O(i)LDA,THF,-78c
(ii)HCOH,THF,-105c
R3
R1
R2
NPh
OH
O
HO
(42)(43)
O
CO2H
R4
R5
(i)Ph3,DIAD,ArOH
(ii)4M H2SO4/Dioxane
reflux
R2
R1
R3
(i)SOCl2/toluenereflux
(ii)SnCl2/CH2Cl2,rt
R4O
R4
R5
R2
R1
R3
R4
AC
BO
(44)(45)
1.3.6.4. Pterocarpan
Despite the identification of the first 6a-hydroxypterocarpan, (+)-pisatin, in
196022, synthetic protocols to these potent phytoalexins are limited by
lengthy multistep routes and a lack of diversity as far as phenolic
hydroxylation patterns are concerned. These confinements are so restrictive
that only two 6a-hydroxypterocarpans, i.e., pisatin and variabilin, have been
synthesized23 the results reported for the stereo selective aldol condensation
between methyl Ketones and aldehydes employing diisopropylethylamine
and chiral boron triflates2iv,25 prompted the investigation for a more direct
synthetic approach to address for the issue of stereo- control at C-6a and C-
11a of the pterocarpan framework and lability and/or stability of protecting
groups under certain reaction conditions. This protocol included
methoxymethyl protection of the benzaldehydes and phenyl acetates as t-
butyl dimethylsilyl (TBDMS) ethers (stable under acidic conditions).
.
R1
R2
OBn
O
TIN/HCLO4
MeOH/rf
(i)H2/Pt/acetone/
rf
(ii)TBDMSCl/imidazol/DMF/rf
OMe
R1
R2
OBn R1
R2
OMe
O O
OTBDMS
(46) (47)(48)
The subsequent condensation between the ester enolates and the benz
aldehydes afforded the 2, 3-diaryl-3-hydroxypropanoates in moderate to
good yields. Cleavage of the silyl ethers using tetrabutylammonium fluoride
(TBAF) on silica26 gave 4-benzylsulfanyl-2`-hydroxyisoflavans, which were
converted to the 6a, 11a-cis-pterocarpans in yields of 39–82% using the
thiophilic Lewis acids,dimethyl (methylthio) sulfonium tetra-fluoroborate
(DMTSF)or silver trifluoromethanesulfonate (CF3SO3Ag)27.
R1
R2
O
OTBDMS
LDA/Et2O
-78c-0c+
R3
H
OMOM
O
R3
OH
MOMO
OOMe
OTBDMS
R2
R1
OMe
R3
OH SBN
OOTBDMS
R2
R1
BnSH/SnCl4/
CH2Cl2/0c
LiAlH4/Et2O
rf
R3
SBN
OTBDMS
R2
R1
OHOHOMe
R3O
SBN
R1
R2
OTBDMS
PPh3/DEAD
rf
R2O
R1
OTBDMS
SBN
TBAF(silica)THF
rf
R2O
R1
OH
SBN
R2O
O
R1
1
2
4
6
7
8
10
11
11a
6a
AgOTf or DMTSF
CH2Cl2/ 0c
1.4. Isolation and identification of flavonoids
Flavonoids and their conjugates form a very large group of natural products.
They are found in many plant tissues, where they are present inside the cells
or on the surfaces of different plant organs.
Flavonoid glycosides are frequently acylated with aliphatic or aromatic
acids. These derivatives are thermally labile and their isolation and further
purification without partial degradation is difficult.
In the plant kingdom, different plant families have characteristic patterns of
flavonoids and their conjugates. All these compounds play important
biochemical and physiological roles in the various cell types or organs (seed,
root,green part, and fruit) where they accumulate28,29,30. Different classes of
flavonoids and their conjugates have numerous functions during the
interactions of plant with the environment.
The identification and structural characterization of flavonoids and their
conjugates isolated from plant material, as single compounds or as part of
mixtures of structurally similar natural products, create some problems due
to the presence of isomeric forms of flavonoid aglycones and their patterns
of glycosylation. A number of analytical methods are used for the
characterization of flavonoids. In many cases nuclear magnetic resonance
(NMR) analyses (H1 and C13) are necessary for the unambiguous
identification of unknown compounds; other instrumental methods(mass
spectrometry, UV and IR spectrophotometry) applied for the identification
of organic compounds fail to provide the information necessary to answer
all the structural questions.
The utilization of various strategies is dependent on the origin of the
biological material from which the target natural products are to be extracted
(plant or animal tissue or body fluids)31.
The utilization of dried plant material for extraction may cause a substantial
decrease in the yield of flavonoid conjugates. Acylated flavonoid glycosides
are especially labile at elevated temperatures and are frequently thermally
degraded during the process of drying plant tissues.
Free flavonoid aglycones exuded by plant tissues (leaf or root) may be
washed from the surface with nonpolar solvents, such as methylene chloride,
ethyl ether, orethyl acetate. However, more polar glycosidic conjugates
dissolve in polar solvents (methanol and ethanol), and these organic solvents
are applied for extraction procedures in Soxhlet apparatus. Mixtures of
alcohol and water in different ratios are applied for the extraction of
flavonoids and their conjugates from solid biological material (plant or
animal tissues and different food products).
The extraction efficiency may be enhanced by the application of
ultrasonication32 or pressurized liquid extraction (PLE), a procedure
performed at elevated temperature ranging from 60oC to 200oC33. However,
the temperature conditions during the extraction procedures have to be
carefully adjusted because of the possibility of thermal degradation of the
flavonoid derivatives.
The choice of the extraction procedure for obtaining flavonoid conjugates
from biological material is very important and depends on the goals of the
conducted research. The evaluation of the spatial distribution of target
compounds on the organ, tissue, cellular, or even subcellular level is of
special interest in some projects.
Multistep chromatographic methods are necessary for the isolation of
individual components from plant extracts containing new uncharacterized
compounds. Various stationary phases are used in column chromatography
including polyamide, sephadex LH-20, and different types of silica gels.
1.4.1 Preparation of body fluids
For the isolation of flavonoids and their derivatives from liquid samples like
beverages (fruit juice) and physiological fluids (blood or urine), two
different approaches are usually applied. The first one is based on liquid–
liquid extraction and the second one on solid-phase extraction of target
natural products mainly on RP C-18 silica gel cartridges.
All physicochemical methods applied in the field of organic chemistry are
useful for structural characterization or identification of individual
flavonoids and their conjugates. The separation approaches mentioned above
may be considered in different ways. The first one is directed toward the
analysis of single compounds obtained after exhaustive isolation and
purification procedures. The method of choice in this approach is H1 and C13
NMR this technique depends on the intensity of the interactions between
different atoms within a molecule placed in a high-intensity magnetic field.
Different NMR experiments have been developed to achieve information
concerning chemical structure of the studied molecule on this basis.
Particularly useful are methods enabling recording of two-dimensional
spectra showing homonuclear interactions [correlation spectroscopy (COSY)
and nuclear overhauser effect spectroscopy (NOESY)] as well as
heteronuclear [heteronuclear single quantum correlation (HSQC) and
heteronuclear multiple bond correlation (HMBC)] to facilitate the
acquisition of all the structural information about an aglycones and the
corresponding sugar substitution.
The NMR spectrometers may be connected on line to liquid chromatographs
(LC-NMR), giving a powerful tool to study mixtures of natural compounds
present in complex samples.
The variety of MS techniques being available in laboratories is a reason that
this technique has a wide range of scientific or practical applications in
biological and medical disciplines.
Important structural data also can be obtained from mass spectra registered
on different types of mass spectrometers (MS). The application of ultraviolet
and infrared spectrophotometers may give valuable information about
specific compounds.
MS applied for the analysis of organic compounds utilize different
ionization methods and may be equipped with different types of analyzers.
In addition, these instruments may be combined with GC/LC or capillary
electrophoresis (CE) apparatus.
1.4.2.. Nuclear magnetic resonance
NMR is a well-established and the most commonly used method for natural
product structure analysis. The studies of flavonoid structures using H1-
NMR were initiated in 1960s34 and along with C13-NMR have become the
method of choice for the structure elucidation of these compounds.
The chemical shifts and multiplicity of signals corresponding to particular
atoms and their coupling with other atoms within the molecule allow for
easy identification of the aglycones structure, the pattern of glycosylation,
and the identity of the sugar moieties present. The literature of this topic is
abundant and rapidly growing35.
1.4.3. Mass spectrometry
Mass spectrometry is a very sensitive analytical method used to
identify flavonoid conjugates or to perform partial structural characterization
using microgram amounts of sample36. Significant structural data can be
obtained from less than 1 mg of the analyzed compound when different MS
techniques are used in combination with chemical derivatization of the
characterized compounds37,75 .
The ionization methods may be divided into two groups differing with
respect to the amount of energy transferred to the molecule during the
ionization process. Electron ionization (EI) belongs to the first group. The
transfer of energy occurs during the interaction of electrons with the
molecule in the vapor state; it may cause the cleavage of chemical bonds and
fragmentation of the molecule, which is characteristic for the analyzed
compound. Other ionization methods deliver lower energy to the studied
molecules during the protonation (positive ion mode) or DE protonation
(negative ion mode) processes. In both cases, the absorbed energy is too low
to cause intense fragmentation. In this situation, techniques of collision-
induced dissociation with tandem MS (CIDMS/MS) have to be applied for
the structural characterization of compounds .The fragmentation
mechanisms are different during high- and low energy collisions used in
electromagnetic or quadruple and ion trap analyzers.
1.5. Nutraceutical flavonoids
“Nutraceutical” is a term coined in 1979 by Stephen DeFelice38. It is
defined “as a food or parts of food that provide medical or health benefits,
including the prevention and treatment of disease.” Subsequently, several
other terms (medical food, functional food, and nutritional supplements)
were used. A nutraceutical is any nontoxic food extract supplement that has
scientifically proven health benefits forboth the treatment and prevention of
disease39 nutraceutical may range from isolated nutrients, dietary
supplements, and diets to genetically engineered “designer” food, herbal
products, and processed products,such as cereals, soups, and beverages. The
increasing interest in nutraceutical reflects the fact that consumers hear
about epidemiological studies indicating that a specific diet or component of
the diet is associated with a lower risk for a certain disease.The major active
nutraceutical ingredients in plants are flavonoids.
The flavonoids are a group of organic molecules ubiquitously distributed in
vascular plants. Approximately 2000 individual members of the flavonoids
group of compounds have been described. As is typical for phenolic
compounds, they can act as potent antioxidants and metal chelators. They
also appear to be effective in cancer. Overall, several of these flavonoids
appear to be effective anticancer promoters and cancer chemo-preventive
agents.
1.6. Flavonoids as antioxidents
Diets high in flavonoids, fruits, and vegetables are protective against a
variety of diseases, particularly cardiovascular disease and some types of
cancer40 Antioxidants and dietary fiber are believed to be the principal
nutrients responsible for these protective effects. Reactive oxygen species
(ROS) are formed in vivo during normal aerobic metabolism and can cause
damage to DNA, proteins, and lipids, despite the natural antioxidant defense
system of all organisms41 ROS contribute to cellular aging42 mutagenesis43
carcinogenesis44 and coronary heart disease45 possibly through the
destabilization of membranes43 DNA damage, and oxidation of low-density
lipoprotein (LDL). Many in vitro studies have demonstrated the potent
peroxyl radical scavenging abilities of flavonoids, which contribute to
inhibiting lipid peroxidation and oxidation of LDL46. Since oxidation of
LDL is implicated in the pathogenesis of coronary heart diseases47 through
its ability to decrease the susceptibility of LDL to oxidation, a number of
researches have undertaken investigations examining the activity of dietary
agents rich in flavonoids in inhibiting LDL oxidation48.
1.6.1. Reactive nitrogen species and inducible nitric oxide synthase
Reactive nitrogen species (RNS) also appear to contribute to the pathology
of cardiovascular diseases. Nitrogen oxide is one RNS- produced by the
action of nitric oxidesynthase in endothelial cells, neurons, and other cell
types. At the sites of inflammation, inducible nitric oxide synthase (iNOS) is
also augmented, and nitric oxide synthesis is further activated. Peroxynitrite,
a potent oxidant generated by the reaction of nitric oxide (NO) with
superoxide in the vascular endothelium, induces LDL oxidation49 and
proinflammatory cytokine-mediated myocardial dysfunction50. Another
potential source of RNS is derived from dietary nitrite,which reacts with the
acidic gastric juice to produce nitrous acid, which decomposes to oxides of
nitrogen. Nitrous acid and its products are able to nitrosate
amines,deaminate DNA bases, and nitrate aromatic compounds including
tyrosine. Several flavonoids and phenolic compounds, including the
epicatechin/gallate family of flavonol, are powerful inhibitors of nitrous
acid-dependent nitration and DNA deamination in vitro51.
1.6.2. Protective effects of flavonoids
The protective effects of flavonoids in biological systems are ascribed to
their capacity to transfer free radical electrons, chelate metal catalysts52
activate antioxidant enzymes53 reduce alpha-tocopherolradicals and inhibit
oxidases54. Green tea is a rich source of flavonoids, primarily catechins and
flavonol. In black tea, as aconsequence of the fermentation process,catechins
are converted to complex condensation products.the. Oral feeding of green
tea leaves to rats resulted in enhanced SOD activity in serum and catalase
activity in liver and an increased concentration of glutathione in the liver55.
Also it was found that theaflavins inhibit xanthine oxidase (XO) and act as
scavengers of super oxides. Theaflavins 3, 3`-digallate (TF-3) inhibited the
superoxide production in HL-60 cells. Therefore, the anti oxidative activity
of tea polyphenols may be due not only to their ability to scavenge super
oxides, but also because of their ability to block XO and relative oxidative
signal transducers56. Other flavonoids such as quercetin,kaempferol,
myristin, apigenin, and leuteolin also have anti oxidative activity in many in
vitro studies57.
1.7- Flavonoids as anticarcinogenesis
Studies on cancer prevention have assessed the impact of a wide variety of
flavonoids and a selected few isoflavones for their efficacy in inhibiting
cancer in a number of animal models. These studies demonstrated that
flavonoids inhibit carcinogenesis in vitro and substantial evidence indicates
that they also do so in vivo58. Flavonoids may inhibit carcinogenesis by
affecting the molecular events in the initiation, promotion, and progression
stages. Animal studies and investigations using different cellular models
suggested that certain flavonoids could inhibit tumor initiation as well as
tumor progression59.
Dietary quercetin inhibited DMBA-induced carcinogenesis in hamster
buccal pouch60 and in rat mammary gland61 when given during the initiation
stage. Quercetin also inhibited DEN- induced lung tumor genesis in mice62
In a medium-term multiorgancarcinogenesis model in rats, quercetin (1% in
the diet) inhibited tumor promotion in the small intestine Feeding rats with
quercetin during either the initiation or promotion stage, inhibited 4-NQO-
induced carcinoma formation in the tongue. Siess and co-workers
investigated the effects of feeding rats with flavone, flavone, tangeretin, and
quercetin on two steps of aflatoxin B1 (AFB1)-induced
hepatocarcinogenesis (initiation and promotion) and found that flavones, and
tangeretin administered through the initiation period decreased cancer risk.
Quercetin decreased oxidative stress-induced neuronal cell membrane
damage more than vitamin C. These results suggest that quercetin, in
addition to many other biological benefits, contributes significantly to the
protective effects of neuronal cells from oxidative stress-induced
neurotoxicity,such as Alzheimer’s disease63. On the other hand, the
suppressive effects of flavones, such as chrysin and a pigenin, on the
expression of the high affinity IgE receptor which plays a central role in the
IgE-mediated allergic response64 has been demonstrate.
Genistein and daidzein (isoflavones derived from soybeans) have been
shown to inhibit the development of both hormone- and non-hormone-
related cancers,including mouse models of breast, prostate, and skin cancer.
Treatment of mice with 100–500 mg genistein/kg diet reduced the incidence
of advanced-stageprostate tumors, in a dose-dependent manner65. A high
isoflavone diet also was shown to inhibit methylnitrosourea-induced prostate
tumor in Wistar rats66. Topically applied genistein reduces the incidence and
multiplicity of skin tumors in the DMBA-initiated and TPA-promoted
multiplicity of skin mouse model by 20% and 50%, respectively66. In the
UVB light-induced complete carcinogenesis model, topical pretreatment of
SKH-1 mice with genistein significantly reduced the formation of H2O2 and
8-hydroxy-2’-deoxyguanosine66.
The anti carcinogenesis effects of green tea, and black tea extracts on
various organs and animal model have been reported67. Studies showed that
green tea polyphenols have a potent inhibitory effect on skin tumorigenicity
in Sensor mice67. A mixture of theaflavin-3-gallate, theaflavin-3′-gallate, and
theaflavin-3, 3`-digallate) reduced NNK-induced lung tumor multiplicity
and volume in A/J mice. These findings are interesting, given the extremely
poor bioavailability of theaflavins, and may suggest that the theaflavins are
metabolized to a more bioavailable active metabolit
In conclusion intensive epidemiological studies have shown consistently that
regular consumption of fruits and vegetables is associated with reduced risk
of chronic diseases such as cancer and cardiovascular disease68. However,
the individual antioxidants of these foods studied in clinical trials, including
β-carotene,vitamin C, and vitamin E, do not appear to have consistent
preventive effects comparable to the observed health benefits of diets rich in
fruits and vegetables69. It has been reported that fresh apples have potent
antioxidant activity; and whole apple extracts inhibit the growth of colon and
liver cancer cells in vitro in a dose dependent manner suggesting that
natural phytochemicals in fresh fruits could be more effective than a dietary
supplement. Apples are commonly consumed and are the major contributors
of phytochemicals in human diets. Apple extracts exhibit strong antioxidant
and ant proliferative activities and the major part of total antioxidant activity
is from the combination of phytochemicals.
Phytochemicals, including phenolic and flavonoids, are likely to be the
bioactive compounds contributing to the benefits of apple. Recent studies
have demonstrated that whole apple extracts prevent mammary cancer in rat
models in a dose-dependent manner at doses comparable to human
consumption of one, three,and six apples per day70.Thus,consumption of
apples may be an effective strategy for cancer chemoprevention.
Chemo-preventive studies have demonstrated that the mechanisms of action
of phytochemicals and nutraceutical in the prevention of cancer go beyond
the antioxidant activity scavenging of free radicals; regulation of gene
expression in cell proliferation, oncogenes, and tumor suppressor genes;
induction of cell cycle asrrestand apoptosis; modulation of enzyme activity
in detoxification, oxidation, and reduction; stimulation of the immune
system; and regulation of hormone metabolism. It is a general theme that the
additive and synergistic effects of phytochemicals and nutraceutical in fruits
and vegetables are responsive for their potent antioxidant and anticancer
activities and that the benefit of a diet rich in fruits and vegetables is
attributed to the complex mixture of phytochemicals and nutraceutical
present in whole foods71.
Recent development in the molecular mechanisms of signal transduction
pathway in various cell systems has provided a strong basis for performing
thesynergistic effects of phytochemicals and nutraceutical in whole foods
that have been ingested by the host. Along this aspect the cancerchemo-
prevention and anti obesity effects of tea and tea polyphenols might
accomplish this through blocking the signal transduction pathways in the
target cells72,73.
1.8-Aim of this work
This work was aimed:
- To screen Acacia nilotica for different phytochemicals.
- To extract the flavonoids from pods of Acacia nilotica.
- To isolate the major flavonoid from the pods of Acacia nilotica.
- To conduct spectral studies (IR and UV) on the isolated component.
1.9-Acacia nilotica
Kingdom: Plantae
Family: Fabaceae
Genus: Vachellia or Acacia
Species: Acacia nilotica
Acacia nilotica tree
Acacia nilotica – flowers
Acacia nilotica –seeds; pods
Acacia nilotica – leaves ,flowers, seed pod
Acacia nilotica commonly known as Egyptian Acacia , is a thorny tree up to
15m in height that produces pods with a characteristic beaded necklace
appearance. The name nilotica originates from its continuous presence in
the Nile vallies where it had been used for tanning and dyeing , as a source
of water – and insect- resistant wood, as a fodder for livestock and in folk
medicine. Different parts of the tree have been used as antiameobic ,
antispasmodic, antidiarrheal and hypotensive. The pods in particular are
used for treatment for fevers, diarrhea, diabetes and skin diseases74.
Phytochemical studies of the aerial parts of the plant resulted in the
identification of a variety phenolic constituents , among which catechin
derivatives were identified. The compounds have a wide range of biological
activities , in particular antioxidant, anticarcinogenic and anti-inflammatory
activities.
3-Results and Discussion
Flavonoids are widely distributed in plants. Flavonoid
compounds are used with considerable interest . They were
reported to have antiviral, anti-allergic, anti-inflammatory, anti-
tumer , anti-malarial and antioxidant activities75,76. Also flavonoids
have important economic values. The diverse properties of these
interesting compounds encouraged us to investigate these
compounds.
3.1-Phytochemical screening.
Phytochemical screening of alcoholic extract of Acacia nilotica
revealed the presence of tannins, glycosides, alkaloids, flavonoids
and steroids. Saponin and terpens were not detected (see table 3.1).
Table (3.1) : Phytochemical screening of Acacia nilotica
Tannins +
Flavonoids +
Alkaloids +
Steroids _
Glycosides +
3.2- Extraction of flavonoids from plant material
Powdered air-dried pods of Acacia nilotica were extracted with
95% ethanol at ambient temperature for 48 hours. Evaporation of
the solvent under reduced pressure gave a crude product which was
applied on Whatman No. 3 sheets. However, the solvent system
that gave optimum separation for flavonoids was acetic acid
(10%) . After the usual work a pure component – compound I was
isolated.
3.3-Identification of compound I.
The IR spectrum of compound I(Fig.1) showed ν(KBr) 662(C-
H, Ar. bending),1070(C-O),1450,1460(C=C ,Ar.),1730(C=O)
,2863,2929(C-H, Aliph.) and 3396cm-1(OH).
Fig.1:The IR spectrum of compound I
The appearance of a carbonyl stretching in the IR spectrum suggests
that compound 1 could be: a flavone , flavonol, chalcone ,aurone,
flavanone, isoflavone, dihydrochalcone or dihydroflavonol. Flavans and
anthocyanins are ruled out since they are devoid of a carbonyl function 1.
Flavone Flavonol
O
O O
O
OH
Chalcone Aurone
Flavanone Isoflavone
Dihydroflavonol Dihydrochalcone
In their UV spectra, most flavonoids show two absorption
bands; band I and II. Band I is associated with the absorption of
the cinnamoyl system, while band II is considered to originate
from the benzoyl system. Flavones, flavonols, chalcones and
aurones give band I and band II, due to conjugation between
benzoyl and cinnamoyl chromophores .
O
O
O
O
O
O
O
O
O
OH
O
OH
Table (3.2) shows the UV absorption of flavones, flavonols,
chalcones and aurones. Flavonols , unlike flavones , possess a 3-
OH functional group. Consequently their UV absorption differ
from that of flavones. Chalcones are characterized by a dominant
band I absorption. In aurones band I is manifested above 390nm.
Such absorption may distinguish them from chalcones.
Table (3.2) : The UV absorption of flavones, flavonols, chalcones and aurones
Flavonoid class Band I Band II
Flavones 330-350 250-270
Flavonols 350-390 250-280
Chalcones 365-390 240-260
Aurones 390-430 240-270
On the other hand , isoflavones ,dihydroflavonols,
dihydrochalcones and flavanones give only band II due to loss of
conjugation between the carbonyl function and ring B.
In the UV compound I absorbs (Fig.2) at λmax(MeOH)
298,400 nm . Thus compound I is probably an aurone (Table 3.2).
Considerable structural features have also been obtained using
UV shift reagents: sodium methoxide,sodium acetate, aluminum
chloride and boric acid/ sodium acetate. These reagents produce
shifts in the UV absorption maxima in accordance with the
location of the various hydroxyl functions in the flavonoid
molecule .
.
Fig..2 UV spectrum of compound I
Sodium methoxide is a strong base and is used as a diagnostic
reagent1 for C3-OH and C4`-OH. In both cases it affords
bathochromic shifts . However , in case of 4`- hydroxylation the
shift is accompanied with decrease in intensity.
The sodium methoxide spectrum of compound I (Fig.3) revealed
a 20 nm bathochromic shift without decrease in intensity indicating
a 4`-hydroxylation.
Fig.3:Sodium methoxide spectrum of compound I
The shift reagent Sodium acetate is a weaker base than NaOMe,
and as such ionizes only the more acidic hydroxyl functions. It is a
diagnostic reagent for detection of 7-hydroxyl group1 .The sodium
acetate spectrum of compound I (Fig.4) did not reveal any
bathochromic shift in band II . This indicates absence of a free 7-
OH .
Fig.4: The sodium acetate spectrum of compound I
Aluminium chloride is another useful shift reagent. It forms
acid-stable chelates with 3-OH and 4- keto function or 5-OH and
4- keto function .It also forms chelates with any catechol systems
in ring A or B . However , the ortho- dihydroxy system, unlike the
3-OH and 5-OH complexes, afford acid-labile complexes2. The
aluminium chloride spectrum of compound I showed a 20nm
bathochromic shift (Fig.5) in band I. was observed in the
aluminium chloride the spectrum (Fig.5). Since the spectrum was
acid – stable then this shift reagent suggests (Fig. 6) a free 5-OH2.
Fig. 5: Aluminium chloride spectrum of compound I
Fig. 6: Aluminium chloride /HCl spectrum of compound I
On the basis of the above cumulative data compound I is a 5,4`-
dihydroxyaurone :
O
OH
HO
O
Conclusion and Recommendations
Using solvent extraction and paper chromatography a 5,4`- dihydroxyaurone
was isolated from the pods of Acacia nilotica.
Recommendation:
The structure of the isolated component may fully be elucidated by other
spectral tools (Proton Nuclear Magnetic Resonance ,Carbon-13 Nuclear
Magnetic Resonance , Heteronuclear Multiple Bond Correlation ,
Heteronuclear Single Quantum Coherence and Mass Spectrometry).
Also the isolated component may be screened for its antimalarial antiulcer
and anti-microbial potential.
The miner components in the pods of this plant may also be isolated and
their structure elucidated in a future study.
3-Results and Discussion
Flavonoids are widely distributed in plants. Flavonoid
compounds are used with considerable interest . They were
reported to have antiviral, anti-allergic, anti-inflammatory, anti-
tumer , anti-malarial and antioxidant activities75,76. Also flavonoids
have important economic values. The diverse properties of these
interesting compounds encouraged us to investigate these
compounds.
3.1-Phytochemical screening.
Phytochemical screening of alcoholic extract of Acacia nilotica
revealed the presence of tannins, glycosides, alkaloids, flavonoids
and steroids. Saponin and terpens were not detected (see table 3.1).
Table (3.1) : Phytochemical screening of Acacia nilotica
Tannins +
Flavonoids +
Alkaloids +
Steroids _
Glycosides +
3.2- Extraction of flavonoids from plant material
Powdered air-dried pods of Acacia nilotica were extracted with
95% ethanol at ambient temperature for 48 hours. Evaporation of
the solvent under reduced pressure gave a crude product which was
applied on Whatman No. 3 sheets. However, the solvent system
that gave optimum separation for flavonoids was acetic acid
(10%) . After the usual work a pure component – compound I was
isolated.
3.3-Identification of compound I.
The IR spectrum of compound I(Fig.1) showed ν(KBr) 662(C-
H, Ar. bending),1070(C-O),1450,1460(C=C ,Ar.),1730(C=O)
,2863,2929(C-H, Aliph.) and 3396cm-1(OH).
Fig.1:The IR spectrum of compound I
The appearance of a carbonyl stretching in the IR spectrum suggests
that compound 1 could be: a flavone , flavonol, chalcone ,aurone,
flavanone, isoflavone, dihydrochalcone or dihydroflavonol. Flavans and
anthocyanins are ruled out since they are devoid of a carbonyl function 1.
Flavone Flavonol
O
O O
O
OH
Chalcone Aurone
Flavanone Isoflavone
Dihydroflavonol Dihydrochalcone
In their UV spectra, most flavonoids show two absorption
bands; band I and II. Band I is associated with the absorption of
the cinnamoyl system, while band II is considered to originate
from the benzoyl system. Flavones, flavonols, chalcones and
aurones give band I and band II, due to conjugation between
benzoyl and cinnamoyl chromophores .
O
O
O
O
O
O
O
O
O
OH
O
OH
Table (3.2) shows the UV absorption of flavones, flavonols,
chalcones and aurones. Flavonols , unlike flavones , possess a 3-
OH functional group. Consequently their UV absorption differ
from that of flavones. Chalcones are characterized by a dominant
band I absorption. In aurones band I is manifested above 390nm.
Such absorption may distinguish them from chalcones.
Table (3.2) : The UV absorption of flavones, flavonols, chalcones and aurones
Flavonoid class Band I Band II
Flavones 330-350 250-270
Flavonols 350-390 250-280
Chalcones 365-390 240-260
Aurones 390-430 240-270
On the other hand , isoflavones ,dihydroflavonols,
dihydrochalcones and flavanones give only band II due to loss of
conjugation between the carbonyl function and ring B.
In the UV compound I absorbs (Fig.2) at λmax(MeOH)
298,400 nm . Thus compound I is probably an aurone (Table 3.2).
Considerable structural features have also been obtained using
UV shift reagents: sodium methoxide,sodium acetate, aluminum
chloride and boric acid/ sodium acetate. These reagents produce
shifts in the UV absorption maxima in accordance with the
location of the various hydroxyl functions in the flavonoid
molecule .
.
Fig..2 UV spectrum of compound I
Sodium methoxide is a strong base and is used as a diagnostic
reagent1 for C3-OH and C4`-OH. In both cases it affords
bathochromic shifts . However , in case of 4`- hydroxylation the
shift is accompanied with decrease in intensity.
The sodium methoxide spectrum of compound I (Fig.3) revealed
a 20 nm bathochromic shift without decrease in intensity indicating
a 4`-hydroxylation.
Fig.3:Sodium methoxide spectrum of compound I
The shift reagent Sodium acetate is a weaker base than NaOMe,
and as such ionizes only the more acidic hydroxyl functions. It is a
diagnostic reagent for detection of 7-hydroxyl group1 .The sodium
acetate spectrum of compound I (Fig.4) did not reveal any
bathochromic shift in band II . This indicates absence of a free 7-
OH .
Fig.4: The sodium acetate spectrum of compound I
Aluminium chloride is another useful shift reagent. It forms
acid-stable chelates with 3-OH and 4- keto function or 5-OH and
4- keto function .It also forms chelates with any catechol systems
in ring A or B . However , the ortho- dihydroxy system, unlike the
3-OH and 5-OH complexes, afford acid-labile complexes2. The
aluminium chloride spectrum of compound I showed a 20nm
bathochromic shift (Fig.5) in band I. was observed in the
aluminium chloride the spectrum (Fig.5). Since the spectrum was
acid – stable then this shift reagent suggests (Fig. 6) a free 5-OH2.
Fig. 5: Aluminium chloride spectrum of compound I
Fig. 6: Aluminium chloride /HCl spectrum of compound I
On the basis of the above cumulative data compound I is a 5,4`-
dihydroxyaurone :
O
OH
HO
O
Conclusion and Recommendations
Using solvent extraction and paper chromatography a 5,4`- dihydroxyaurone
was isolated from the pods of Acacia nilotica.
Recommendation:
The structure of the isolated component may fully be elucidated by other
spectral tools (Proton Nuclear Magnetic Resonance ,Carbon-13 Nuclear
Magnetic Resonance , Heteronuclear Multiple Bond Correlation ,
Heteronuclear Single Quantum Coherence and Mass Spectrometry).
Also the isolated component may be screened for its antimalarial antiulcer
and anti-microbial potential.
The miner components in the pods of this plant may also be isolated and
their structure elucidated in a future study.
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