Isolation and Identification of Flavonoids from leaves of BauhiniarufescensLam.

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Isolation and Identification of Flavonoids from leaves of BauhiniarufescensLam.
Mustafa S.Koya1, Nada A. Elamin2. , Syed Z. Idid3
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,
فصل الفلافونويدات من أوراق نبات الكلكل
والتعرف على تركيبها الكيميائي
مستخلص الدراسة:
أجريتتهذهتتلدذاسة اغتت ذصلتترفذفوتتدذاسمنفأورتتةابذتتت ذأ ا ذو تت بذذ) Bauhinia rufescensLam (ذذ"ذاسكلكتتد"استتليذيمي تت ذسلع ئلتت ذاس لأسرتت ذ استت ذأ ضتتاذاح تتاذا ستت ذ
سليعرفذعلىذاحكأو بذاسكر ر ئر ذاسمع س ذك ض دابذأك ةةذصأوهت ذا ذكثترذ فترةذ ذذ
أ ا ذاسم بذتحهذاسة اغ .ذ
اغتتيتةتهذتتتلي بذاوك تتت ذ تتتنبذاإثرتتتدذ احثتتت وألاذ غتتيتن ذاحكأوتتت بذ
اسكر ر ئر . تمهذع لر ذفودذاحكأو بذاسكر ر ئرت ذص غتيتةاتذنلمرت ذكر تيأفرافرت ذذ
اسط ل ذاسرقرل ذ كر تيأفرافر ذاسع أد.ذ
تمذالحوألاذعلىذترك ينذذ حةدذنركر ه ذاسكر ر ئ ذص غيتةاتذنلمر بذطرت ذذ
ا شتتع ذفتتتأ ذاس مم تتتار ذ طرتت ذا شتتع ذالح تتترااذ تطر فرتتت ذاسكيلتتت ذ استتتروينذاسمتتتأ يذ
احلمطر .ذ
أ ضتت هذاسميتت ئ ذأذ ذاحتترك ينذاحموتتأسينذه تت ذتتت ذاحرك تت بذاسمرمأسرتت ذ:ذحمتت ذ
اسكلأ جمتتت ذذ) Chlorobenic ذحمتتتت ذفرمتتتتأس (ذ تركتتتترذاستتتتر نينذ) Rutin (ذهتتتتأذتتتتت ذ
اسمنفأورةاب.ذ

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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

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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

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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.

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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 40ᵒC. The obtained dry extract (18.4g) was kept in fridge for further analysis.
2- 3General experimental procedure:-
Proton and 13C-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

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(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. 1H NMR (400Hz,CD3OD,
) spectral data of compoud1are displayed in details on table(1).

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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

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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) 13CNMR (DMSO-d6,125MHz).
Table 2.1H and 13C 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

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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

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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 13C-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

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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.

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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-

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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.

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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.
References
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