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

53

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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