Ethnobotanical Leaflets 12: 245-253. 2008.
Phytochemical Investigation and Pharmocological Studies of the Flowers of Pithecellobium dulce
1Lecturer in Chemistry,
2Lecturer in Bioinformatics, Department of Biotechnology, Manipal Institute of Technology, Manipal University, Manipal-576104, India
2Corresponding Author: Tel: mobile-9895592347
Issued 24 May 2008
To evaluate the effects from the fresh flowers of Pithecellobium dulce (Roxb.) Benth, belonging to the family of Leguminosae subfamily Mimosoideae, a glycoside quercitin has been isolated. The ethyl acetate soluble of P. dulce containing the above glycoside was studied both in silico and in vitro for the anti-inflammatory and anti-bacterial properties. The concatenation of the in silico and in vivo has been done. Results indicated the activity of this flavonol glycoside in the protection of HRBC lysis and against the gram positive micro organisms, thus confirming its anti-inflammatory and anti-bacterial properties.
Pithecellobium dulce (Roxb.) Benth, belonging to the family of Leguminosae, subfamily Mimosoideae, is a small evergreen thorny tree1. The tree is reported to be active against veneral diseases2. The decoction is given as enema3. The seeds contain saponin4. As there is no work on the flowers of P. dulce, the flowers of same have now been examined for their poly-phenolic constituents. The crude extract of the flowers has also been investigated in silico and in vitro for their anti-inflammatory and anti-bacterial properties and our results are presented in this communication.
From the fresh flowers (300g) of P. dulce
collected from Thanjavur district of Tamilnadu,
255,269sh, 370 ;(MeOH+NaOMe):262sh,321,420(dec.);(MeOH+AlCl3):
267,303,458 ;( MeOH+AlCl3+HCl):267,303,351,428;(MeOH+NaOAc):
275,328,390 ;( MeOH+NaOAc+H3BO3):262,303sh, 386;
red color with Mg-HCl5, Olive green color with alc.Fe3+, golden yellow
color with NH3 and NaOH, yellow solution with pale green fluorescence
with conc.H2SO4, yellow under UV/NH3, responded to Horhammer-
flavonol 3-O glycoside and the identity was confirmed by co- and mixed
PC with an authentic sample of quercetin.
The 13C- NMR data of the isolated compound is given as follows:
51) and 122.0(C-61)
The UV and 13C- NMR spectral data were in agreement with the flavonol quercetin.
A solution of the glycoside was hydrolyzed (7%H2SO4, 100 C, 2 hr.) and the aglycone was characterized as quercetin (m.m.p, uv data, Rf, acetate and methyl ether) and the sugar was identified as rhamnose. A quantitative hydrolysis of the same by the Folin-wu’s micro method 22 revealed to be a monoside. The glycoside was thus characterized as quercetin 3-O rhamnoside (quercitrin) and the identity was confirmed by direct comparison with an authentic sample of quercitin. Based on the UV and 13C- NMR spectral data, it is crystal clear that the isolate is flavonol quercetin. The structure of the quercitrin was drawn by using ACD-3D ChemSketch.5 Then we performed the conversion of the drawn chemical structure into SMILES 7 notation, so as to predict biological activity and to find similar chemical compounds. The SMILES notation used for screening similar compounds and biological activity is given below.
To find similar chemical compounds we have screened the compounds from
Quercetin 3-O-rhamnoside (quercitrin)
see figure 1, has been isolated from the fresh flowers of P. dulce. The UV spectrum of the glycoside exhibited two
major absorption peaks at 350 nm (band I) and 256 nm (band II). The band I
absorption of the glycoside is reminiscent of a flavonol
skeleton. A comparison of band I absorption of the glycoside and that of the aglycone revealed that there may be 3-glycosilation in
the flavonol. A bathochromic
shift of 43 nm (band I) in NaOMe confirmed the
presence of a free –OH at C-41 .The AlCl3 spectra (with
and without HCl) showed four absorption peaks to
reveal the presence of a free 5-OH group. It was confirmed by the bathochromic shift of 50 nm on the addition of AlCl3-HCl
in the glycoside. The presence of a free –OH group at C-7 was evident from
the +16 nm (band II) shift on the addition of NaOAc.
The band I absorption in AlCl3
spectrum is 30 nm more than that noticed on addition of AlCl3-HCl.This
is indicative of the existence of an
O- dihydroxyl group in the B-ring. In the 1H-NMR
Figure 1: Quercitrin structure.
The stick model shown in this picture (color by
Thus on the basis of the above mentioned Physical and Chemical
evidences the glycoside obtained from P. dulce
has been characterized as quercetrin. Biological
activity prediction is pivotal in any structure prediction. Based on the UV
and 13C- NMR spectral data, it is crystal clear that the isolate is flavonol quercitrin. We were
much interested to predict its biological activity. So as to predict
structure activity relationship, for that the structure of the quercitrin was drawn by using ACD-3D ChemSketch
v 5.12 5. We have performed in silico
studies like chemical structure similarity search and the Prediction of
Activity Spectra for Substances: PASS 6 prediction was done
by converting the quercetin structure into the
Simplified Molecular Input Line Entry System (SMILES notation) 7
proposed by Dave Weininger (Weininger,
1988). Finally we have found some similar structures in enhanced
Table 1: PREDICTED ACTIVITY
Table: 2. PREDICTED RELIABLE EFFECTS.
Table: 3. PREDICTED MECHANISMS
So from the in silico predicted information, we have decided to test the activity in vitro especially for the membrane permeability inhibitor (Table-1), membrane integrity agonist (Table 1, 2, 3), anti-bacterial effects(Table-2) and anti-inflammatory mechanisms (Table-2 & 3).
Lysosomal enzymes play an important role in
the development of acute and chronic inflammation.9
Increased enzyme activity has been reported in certain types of experimental
inflammation.10 The inhibitory effects of non-steroidal
anti–inflammatory drugs on lysosomal enzymes have
been proposed as an explanation for one of their many mechanisms of actions in
vitro.11 Acidic anti-inflammatory compounds such as
phenyl butazone, Mefenamic
acid and indomethacin have been shown to exert
their beneficial effect by inhibiting the activities of either released lysosomal enzyme or by stabilizing the lysosomal membrane12-14. It has been
reported that the structure of
Table: 4: Stabilization Effect of isolates of P.dulce on the HRBC membrane stabilization against hypotonicity induced haemolysis.
Based on the in vitro Stabilization Effect of isolates of P.dulce on the HRBC membrane stabilization against hypotonicity induced haemolysis, the tabular values are plotted as a graphical representation. (Graph-I)
Graph-I: The in vitro Stabilization Effect of isolates of P.dulce on the HRBC membrane stabilization against hypotonicity induced haemolysis. The concentration of the drug (in mg) used in the protection of HRBC membrane is plotted in the graph.
The crude extract was observed to be effective in stabilizing the HRBC membrane against hypotonicity induced haemolysis and hence would be effective as non steroidal anti-inflammatory compounds in the control of inflammation. With in the experimental range of dosages of (10 to 250 mg /ml) the flavonoid drug exhibited 70% protection at 250 mg dose and at subsequent doses, the protection increases and reached a maximum with a sharp increase at 500 mg. At higher concentrations the activity climbs up showing the anti-inflammatory activity of this flavonoid drug under in vitro experimental conditions dependent upon the concentration of the drug. The membrane is stabilized by the flavonoidal drug at a concentration of 500 mg.
In this investigation , the anti- bacterial activity of the residue of the EtOAc fraction containing the flavonoid glycoside isolated from the flowers of P.dulce have been studied in vitro by Petri-dish method using Staphylococcus aureus a gram positive, Escherichia coli and Salmonella typhii two gram negative as test organisms. Anti-bacterials produce their effect by interfering with one or more vital metabolic pathways in the organism. The object of the treatment with an anti-microbial drug which is higher than the minimal effective concentration and which is maintained at that level until the organisms have been eliminated 17.The extracts of various medicinal plants containing flavonoids have been reported to possess anti- bacterial activity 18.A standard volume (2.5mL) of Mueller-Hinton agar medium that would support the growth of the test organisms was added to sterile Petri–dishes. Solutions of the test compound (EtOAc residue) at six different concentrations viz., 25, 50,100,200,300 and 400 mg/mL in sterile water were prepared. Standards containing streptomycin at concentration of 50,100 and 200 mg/mL and a control containing no drug were prepared. A standard inoculum of a suspension of turbidity equal to a McFarland standard 0.5 of the test organism was added to all Petri–dishes. After inoculation, the plates were incubated at 37C and minimum inhibitory concentration (MIC) is found out after 48 hours of incubation. The number of colonies that grow on this subculture is then counted and compared to the number of CFU/mL (Colony Forming Units) in the original inoculum. In the anti-microbial studies only traces of the growth has been observed at a lower concentration of the drug. The growth of the organism is inhibited with higher concentration.
The authors express their thanks to their Dean, Dr. K .N. Somesekharan, and Dr. D. Venkappayya, Professor, School of Chemical & Bio Technology, SASTRA Deemed University, Thanjavur, Tamilnadu for their keen interest and constant encouragement .They are also grateful to Dr.K.M.Matthew, Rapinat herbarium, St.Josephs college, Tiruchiraapalli for his help in the identification of the plant.
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