Isolation and Identification of Flavonoids from leaves of BauhiniarufescensLam.
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Transcript of Isolation and Identification of Flavonoids from leaves of BauhiniarufescensLam.
53
Isolation and Identification of Flavonoids from leaves of
BauhiniarufescensLam. Mustafa S.Koya
1, Nada A. Elamin
2. , Syed Z. Idid
3
1department of Chemistry, faculty of Education, university of Nyala Sudan
2department of Biology, faculty of Education,University of Nyala, Sudan
3Center for Foundation Studies (Petaling Jaya campus), International Islamic
University Malaysia,
وراق نبات الكلكلأمن اتفصل الفالفونويد
والتعرف على تركيبها الكيميائي
:مستخلص الدراسة
Bauhinia)جريتتهذهتتلدذاسة اغتت ذصلتترفذفوتتدذاسمنفأورتتةابذتتت ذأ ا ذو تت بذذذذذذذأ
rufescensLamضتتاذاح تتاذا ستت ذأسلع ئلتت ذاس لأسرتت ذ استت ذذاستتليذيمي تت "اسكلكتتد(ذذ"ذ
كثترذ فترةذ ذذذك ةةذصأوهت ذا ذأ ذاسمع س ذك ض دابذسليعرفذعلىذاحكأو بذاسكر ر ئر
ذ. ا ذاسم بذحتهذاسة اغ أ
ثرتتتدذ احثتتت وأالذ غتتتيتن ذاحكأوتتت بذاغتتتيتةتهذتتتتلي بذاوك تتت ذ تتتنبذاإ
متهذع لر ذفودذاحكأو بذاسكر ر ئرت ذص غتيتةاتذنلمرت ذكر تيأفرافرت ذذذذذ.اسكر ر ئر
ذ.اسط ل ذاسرقرل ذ كر تيأفرافر ذاسع أد
رت ذذعلىذترك نيذذ حةدذنركر ه ذاسكر ر ئ ذص غيتةاتذنلمر بذطذ متذاحلوأال
شتتتع ذاحل تتترااذ تطر فرتتت ذاسكيلتتت ذ استتترونيذاسمتتتأ يذا شتتتع ذفتتتأ ذاس مم تتتار ذ طرتتت ذا
ذ.احلمطر
محتت ذ:ذ ذاحتترك نيذاحموتتأسنيذه تت ذتتت ذاحرك تت بذاسمرمأسرتت ذذذذأ ضتت هذاسميتت ئ ذأذ
هتتتتأذتتتتت ذ(ذRutin)ذ تركتتتترذاستتتتر نني(ذمحتتتت ذفرمتتتتأس ذChlorobenic)ذاسكلأ جمتتت ذ
ذ.اسمنفأورةاب
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Abstract
The aim of this study was to isolate and identify the flavonoids
from, Bauhinia rufescensLam. Indigenous in the area of study.
Crude extract of leaves from Bauhinia rufescensLam.
family(Fabaceae) was phytochemically investigated. The methanolic
extract was partitioned with different solvents systems by increasing
their polarities ( n- hexane, ethyl acetate, methanol, and 70%
methanol). The compounds were fractionated and isolated from,
methanol and ethyl acetate fractions by using vacuum liquid
chromatography (VLC), silica gel-column chromatography and
preparative thin layer (RP-18F254) chromatographic techniques.
Detection was accomplished with UV. Lamp at =254 nm and
365nm. The structures of the isolated compounds were elucidated by
extensive spectroscopic studies via (1HNMR,
13CNMR, IR,UV. and
MS).The methanolic and ethyl acetate fractionates yield two phenolic
compounds, which were identified aschlorogenic acid and Rutin
(Queercetin-3-O-rutineside).
Keywords:-Flavonoids, Bauhinia rufescens,methanolic
extract,chlorogeni acid, Rutin,
Introduction
Plants have potent biochemicals and have vital components
asphytomedicines (Mashes and Patni, 2008). The tropical rain plants
are biologically and chemically diverse resources, as they synthesize
various chemicals and defense agents against pets, diseases and
53
predators. They are excellent reservoir of medicines and chemicals
leads with which researchers can design and synthesize new drugs
(Ibrahim, 2004).
It is estimated that 80% of the world populations in developing
countries are totally dependent on medicinal plants for their primary
health care (WHO, 2001).More than 3500 plant species were reported
to be used in various human cultures around the world for medicinal
purposes, 11% 0f the 252 drugs considered as basic and essential by
WHO are exclusively of plant origin, and a significant number of
them are synthetic drugs derived from natural precursors (Rates,2001).
Bauhiniais a genus of more than 600 species grows in the
tropical region of the world (Larson, 1974).Bauhinia rufescens Lam.
belonging to the family Legumiosea- caesalipioideae is small
branched shrub or small tree grows up to 8m high with white flowers.
It is found in the entire Sahel and adjacent Sudan zone , from Senegal
and Mauritania across west Africa extending to Sudan, and often
grown as an ornamental plant in villages(Aminu and
Hassanh,2013).Leaves and fruits are applied for the treatment of
diarrhea, dysentery, Jaundice, and diabetes mellitus( Aliyu et al,
2009). Flavonoids such as quercetin and Kaempferitrine have been
isolated from leaves of a Brazilian species of Bauhinia Froficata(
Silva et al,2000).
Flavonoids are natural products widely distributed in the plant
kingdom (Paul et al,2012). They are the most characteristic class of
compounds in the higher plants( Trease and evans,1989).Flavonoids
53
are categorized according to their molecular structures into flavonols,
flavones, flavans, flavanones, isoflavones, catechin,anthocynidin and
chalcones( Ren et al,2003).These compounds appear to play a vital
role in defense against pathogens and predators( Meena and
Panti,2008).They are synthesized from phenylpropanoid and acetate
derived precursors.Flavonoids are important for human beings due to
their antioxidant and radical scavenging effects as well as their potent
estrogenic and anticancer activities(Satio and Fujisawa,2003).
Based on the uses of the leaves and fruits of Bauhinia rufescens
Lam.in folkmedicine for the treatment of diarrhea, dysentery,
Jaundice, and diabetes mellitus, the crude extract of the leaves was
phytochemically investigated by our team in a previous work. The
results revealed that flavonoids were the predominant chemicals.
Thus the main objective of the present study is to isolate and
identify flavonoids indigenous in the leaves of Bauhinia
rufescensLam, with the intention of coming out with a novel
compound.
Materials &Methods
2-1 plant sample collection:-
The leaves of bauhinia rufescens were collected from rural areas
of south Darfur state around Nyala town in July 2010. The plant was
identified by a botanist and a specimen was deposited in botany lab.
University of Nyala Faculty of Education.
53
2-2 Extraction and fractionation of plant material:-
The fresh leaves were collected and air dried for several days. The
air dried plant material (about 1Kg) was ground into powder and
extracted in soxhlet successively using n-hexane, ethyl acetate,
methanol and 70% aqueous ethanol. The ethanolic fraction (35.5g) dry
powder was packed up into silica gel(60F254) column (19x10cm) and
subjected to fractionation by vacuum liquid chromatography (VLC)
technique, n-hexane, EtOAC and EtOH were used as eluents. The
fractions of the three eluents were exposed to evaporation over night.
EtOH and EtOAc fractions were combined based on their TLC
chromatographic similarity, and subject to concentration using rotary
evaporator at 40ᵒ C. The obtained dry extract (18.4g) was kept in
fridge for further analysis.
2- 3 General experimental procedure:-
Proton and 13
C-NMR was recorded using Mercury- 200BB
(400Hz), at the department of plant sciences University of Gifu-
Japan.Uv spectra were measured on UV. Perkin Elmer
Lambda.(Germany).I.R spectra were recorded in KBr discs using
FTIR( Perkin Elymer1600).The mass spectrum were determined
on FAB-MS( VG70SE) Mass electronic U.K London. All
chemicals and reagents were of analytic grade and were obtained
from Fisher Scientific Spring field, Sigma and Merck.
2-4 Isolation and identificationof compounds:-
18.4g of chloroform soluble plant material obtained from VLC was
subjected to a series of open column chromatography on silica gel
04
(Merck60F254, 70-230mesh) using n-hexane, EtoAc and EtOH as
eluents in increasing polarity n- hexane –EtOAc(9:1, 8:2 ,
7:3,1:1.6:4,8:2EtOAc-EtOH( 8:2,6:4,4:6,8:2)monitoring by thin layer
chromate graphy (TLC) the yield fractions were recombined and
repeatedly subjected to column chromatography to get two major sub-
fractions.
Results:
Compound1 was obtained as colorless crystals(25mg,) on silica gel
column chromatography(3x40cm) and eluted with n- hexane –
EtOAc(9:1,8:2, 7:3,1:1.6:4,8:2EtOAc-
EtOH(8:2,6:4,4:6,8:2)V/V,monitored by thin layer
chromatogragraphyTLC,(RP-18F254)Rf -value of which was
determined as0.5 in EtOAc-MeOH(1:1).Its m.p222-224°C
uncorrected. UV spectrum of compound1 gave absorptionλmaxat
223(MeOH) , and IR Vmax (cm-1
, KBr disc): at 3520 (OH), 3362,
3090, 1612 (C=O), 1578, 1436,(aromatic) 1362, 1108 (C-O), 1072,
The FAB-MS spectrum of compound1displayeda molecular ion Peak
at 353m/z [M-H+] and two other fragmentation ions at 289and 153
m/z. 1
H NMR (400Hz,CD3OD, ) spectral data of compoud1are
displayed in details on table(1).
04
Table 1 1H-NMRand
13CNMR signal assignments established for
compound1from B.rufescens leaves. in1 DMSO-d6 (δ= ppm)
Position multiplicity
Coupling constants(Hz)
2 160 7.05 1H,d,J=2Hz,H-2
3 102
4 160
5 109 6.77 1H,dd,J, 8Hz,H-5
6 140 6.95 1H,dd,J,2.8Hz,H-6
7 111 7.56 1H,d,J,16Hz,H-7
8 94.8 6.28 1H, d,J,16Hz,H-8
9 102
10 109
1’ 102
2’ 75.1 2.02-2.11 2H,mH-3
3’ 78.7 5.32 1H,m,H-3’
4’ 71 3.72 1H,dd,J,2.8Hz,H-4’
5’ 77 4.17 1H,m,H-6’
6’ 68 2.16-2.25
Compound2was isolated as yellow amorphous powder by
successive column chromatography of (19.1g) of EtoAc soluble plant
material over silica gel and sephadex LH-20 column , n-hexane,
EtOAc and MeOH gradient in increasing polarity were used as
eluents, and purification was carried out by acetone. The Rf of
compound2 was determined as 0.8 on MeOH and EtOAc(1:1) ;m.p.
189-191°C. (lit., 186-189°C; Hamza et al., 1998) UV (MeOH) )
λmax359 nm (band І) and 257 nm(band ІІ); Two more absorption bands
02
at382 nm and 422 nm were observed upon addition of NaOAc and
NaOMe; FT- IR spectrum of the compound 2 showed (KBr) Vmax
3412 cm-1
(OH str.) , 1656 cm-1
(C=O), 1611 cm-1
(C=C), 1509, 1450
cm-1
(Aromatic group) 1116 cm- 1
(C-O-carbonyl) ; FB/ MSof
compound2 showed m/z 611[M+H]+.and other fragmentation ions at
m/z 465. [M+H-146]+ and m/z 303.2 [M+H-308]+).
The1 H-NMR
13C NMR of compound2 signals are assigned as
in ( Tale 2) 1H NMR (chemical shift δ in ppm, coupling constant J in
Hz) (CD3OD, 600) 13
CNMR (DMSO-d6,125MHz).
Table 2.1H and
13
C NMR signal assignments established for compound2
Atomic
number
DEPT
(ppm)
(ppm)
J (Hz)
Lit. (ppm)
Aglycone
2 C 158.5 157.1
3 C 145.5 134.0
4 C 179.4 178.0
5 C 163.0 161.9
6 CH 99.9 6.20 d.2.2 99.4
7 C 166.1 165.0
8 CH 94.9 6.39 d.2.1 94.3
9 C 149.8 157.3
10 C 105.6 104.5
1’ C 123.1 121.8
2’ CH 123.5 7.76 d.2.1 116.9
3’ C 117.7 145.4
4’ C 159.3 149.1
5’ CH 116 6.87 d.8.3 115.9
05
6’ CH 135.6 7.65 dd.2.12/8.9 122.3
Glucose
1’’ CH 104.7 5.11 d.6.7 101.9
2’’ CH 75.7 74.7
3’’ CH 77.2 4.10-
4.40
76.6
4’’ CH 71.4 70.4
5’’ CH 78.1 77.0
6’’ CH2 68.5 3.4a,3.6b 67.4
Rhamnose
1’’’ CH 102.4 4.52 101.9
2’’’ CH 72.1 71.0
3’’’ CH 72.2 4.10-
4.40
71.2
4’’’ CH 73.9 72.5
5’’’ CH 68.7 68.9
6’’’ CH3 17.9 1.2 d.6.1 18.4
4-Discussion
The structural identification of the obtained compounds was
carried out by interpretation of extensive spectroscopic data and
comparison with the data in the literature and they were found to be
consistent with thedata in the literature. Furthermore, they were
assayed for thin layer chromatographic behavior with various solvent
systems and visualizing reagents and the results were almost the same
as those of the pure authentic samples.
The MeOH&EtOAc extracts were fractionated into n-hexane,
ethyl acetate and methanol through solvent fractionation. The
00
repeated silica gel and sephadex LH-20 column chromatographic of
EtOAc fractions provided two phenolic compounds.
Compound1 gave a strong blue fluorescence under UV-light at
365nm which remained unchanged when sprayed with10% AlCl3
solution in methanol indicating that compound1 is a phenolic acids
member, (Harborneet al., 1999). The FAB-MS spectrum of
compound1displayed a molecular ion Peak at 353m/z [M-H
+] which is
consistent with the molecular formula (C16H18O9). IR spectra of
compound1 showed frequencies at 3520 cm
-1(OH), 2970, 2832cm
-
1indcating the presence of hydroxyl group and C-H in conjugation,
respectively and the absorption peaks at 1650,1612 and 1578cm-1
indicate the presence of carbonyl (C=O), and aromatic ring, peaks at
1362, 1108 were evidence for (C-O). 833 cm-1
for, monosubtituen
benzene.
1 H-NMR &
13C NMR of compound1 showed signals that are
assigned as follows: δ 7.56 (1H, d, J = 16 Hz, H-7), 7.05 (1H, d, J = 2
Hz,. H-2), 6.95 (1H, dd, J= 2, 8 Hz, H-6), 6.77 (1H, d, J = 8 Hz, H-5),
6.28 (1H, d, J = 16 Hz, H-8), 5.32 (1H, m, H-3´), 4.17 (1H, m, H-5´),
3.72 (1H, dd, J = 3, 8 Hz, H-4´), 2.16-2.25 (2H, m, H-6´), 2.02-2.11
(2H, m, H-2´). The 13
C-NMR(125 MHz, in DMSO-d6).spectra
showed carbons at δC160 (C-2); 102 (C-3); 102.3 (C-5); 140 (C-6);
111.2 (C-7); 94.8 (C-8); 102.2 (C-9); 109 (C-10); 102.2 (C-1’),75(C-
2’) 78(C-3’)71(C-4’)77(C-5’)and 68(C-6’).see table(1). These spectral
data indicated that compound 1has a phenolic derivative with
03
cyclohexane skeleton as part of its structure, thuscompound1 was
assumed to be Chlorogenicacid (fig.1)
The FAB-MS analysis in the positive ion mode displayed a
protonated molecule at m/z 611[M+H]+, (calc. for C27H30O16) with
fragmentation showing the departure of two sugar units (ions at m/z
465.0 [M+H-146]+ and m/z 303.2 [M+H-308]+) suggesting the
presence of a flavonol di-glycoside.( Mabry et al., 1976 ).
The UV spectrum of compound 2 showed two major absorption
bands at 359 nm(band І) and 257 nm(band ІІ), which indicated the
presence of flavonol structure( Markham, 1982).Two more absorption
bands at382 nm and 422 nm were observed upon addition of NaOAc
and NaOMe indicating the presence of a free hydroxyl atC-4’ , C-7
and orthodihydroxy in B ring.
03
Bathochromic shift in band (ІІ) with AlCl3 and AlCl3/HCl was an
evidence for presence of free hydroxyl group in positions C-5 and that
the structure might contain adjacent hydroxyl groups (Bacon and
Mabry, 1976)
In the 1H NMR spectrum the two ortho-coupled aromatic protons
at 7.67 (1H, d, J=2.1 Hz) and 7.66 (1H, d, J=2.1 Hz) and the singlet
aromatic proton detected at 6.87 (1H, (d,J=8.3Hz) formed an ABX
system confirmed the di-substitution on the B-ring.
Observation of the two meta-coupled protons at 6.20 (1H, d, J=1.8
Hz, H-6) and 6.39 (1H, d, J=2.2 Hz, H-8) allowed attribution of the
third oxygenation of the A-ring to C-7.The high field signal at
δH1.12ppm which integrated for three protons suggested presence of a
methyl group which is a part of rahmanose sugar unit. Ten proton
signals at sugar region in 1H spectrum of compound2 confirmed the
existence of two sugar moieties.
1 H-NMR &
13C NMR of compound2 showed signals that are
assigned as follows
(calc. for C27H30O16). ions at; MHz): 6.20(1H, d, J=2.2, C6-H),
6.39 (1H, d, J=2.1, C8-H),7.76 (1H, d, J=2.1,C2’-H),6.87 (1H, d
J=8.3,C5’-H),7.65 (1H, dd, J=9,2.1, C6’-H). 5.11 (1H, d, J=7.6,C-
1”H),4.52 (1H, d, J=8.3, C-1”’H), 3.25-3.47 (4H, m, H-2” ,H-3”, H-
4”, H-5”), 3.31-3.25,(4H,m,H-2”’,H-3”’,H-4”’,H-5”’),3.55d (1H, m,
Ha-6”), 3.54 (1H, d, J = 10.5 Hz, Hb-6”) 1.2 (3H,. d, J=6.1,CH3-).
13C NMR (DMSO-d6,125MHz):δC158.5 (C-2), 135.6(C3)
,179.(C4)162.5 (C-5), 99.9 (C-6), 166.0 (C-7), 94.8 (C-8), 159.3 (C-
03
9), 105.6 (C-10), 123.1 (C-1_), 117.6 (C-2_), 145.8 (C-3_), 149.7 (C-
4_), 116.1 (C-5), 123.5 (C-6_), 104.7 (C-1), 75.7 (C-2), 77.2 (C-3),
71.4 (C-4), 78.1 (C-5), 68.6 (C-6), 102.4 (C-1), 72.0 (C-2), 72.2 (C-3),
73.9 (C-4), 69.7 (C-5), 17.9 (C-6).See table(2)
The 1H-NMR and
13C-NMR of compound2 revealed the chemical
shifts of protons and carbons essentially identical with those reported
in the literature for a flavonol glycosiderutin(DerMarderosian,2001).
Based on the above data compound2 was identified as quercetin-
3-O-rutionside which was in agreement with the figure depicted
below.
03
Acid hydrolysis, TLC-analysis and comparison of compound2
with the authentic standards confirmed that the two sugar moieties are
glucose and rahmonse whereas the aglycone is quercetin.
Conclusion
Medicinal plants used in folk medicine may be an interesting and
largely unexplored source for the development of potential new
compounds.Thus it is necessary to isolate the active principle and
characterize their constituents for the benefit of the human beings. It
was our attempt to identify the new compounds in this plant that
revealed two compounds both of them are previously reported,
however to our knowledge it is the first time they are isolated from
Bauhinia rufescensLam.
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