Isolation and Characterization of Flavonoid and Other Compounds from Seeds Extract of Calpurnia Aurea

The study was undertaken to isolate and characterize flavonoid and other compounds from seeds extract of Calpurnia aurea. Three compounds were isolated from this medicinal plant, it were identified as flavonoid compound G1 named: 2-(4-(2-hydroxyethyl)phenyl)-4H-chromen-4-one, compound P2 named: 1-(2-(3-((E)-buta1,3-dienyl) cyclopentyl)ethyl)benzene and compound C3 named: (E)-methyl 14-hydroxy-10-methyltetradec-2enoate. Its structures determinations were based on H, C NMR spectral measurements. In this study, proton and carbon signals were assigned by means of 2D NMR spectral methods for compound P2.


Extraction and isolation
Compound G1, the collected seeds were washed thoroughly in tap water, shade dried and pulverized. Compound G1 was extracted using 30 g of pulverized plant material and was extracted with 180 ml of petroleum ether in a shaker for 24 hours. The solid residue obtained was then treated with ethyl acetate for 24 hours and filtered. The resulting filtrate was concentrated using flash evaporator for complete solvent removal. The freeze dried material was extracted with boiling acetone and the residue was concentrated at atmospheric pressure. This concentrated residue was extracted successively with light petroleum ether and benzene to remove non flavonoid and other matter.

Phytochemical screening tests
Phytochemical screening tests were done to determine the class of compounds present in both crude extract, by following the standard procedures [10,11].  Tannin  -+  6 Steroid + -7 Anthraquinone -+ 8 Phenol -+

Instruments
NMR spectra were recorded on a Bruker Advance instrument (400 MHz and 100 MHz) and with TMS as an internal standard (chemical shifts in δ, ppm). 2D NMR (400 MHz, DMSO-d6) spectra were recorded under standard conditions. The isolated compounds were dissolved in DMSO-d6 and analyzed with 1D NMR (proton 1 H, carbon 13 C), 2D NMR (HMBC and HMQC) and LC-MS.
In the 13 C NMR spectrum, there were seventeen carbon signals and very intense signals at δC128.7 and δC 127.4 each of which almost certainly represents two carbon atoms (C13, 15 and C12, 16). Signals at δC131.7, 124.5, 136.3, 118.7, 158.3 and δC125 assigned for the second aromatic ring. Signal at δC183 showed there was carbonyl group in the structure.
All chemical shift data in the 1D-NMR spectra agreed with the proposed structure for the compound G1: 2-

Characterization of compound P2
Compound P2 was obtained as a black solid from methanol extract. Its molecular formula, C17H22 was determined by negative LC-MS. In the negative LC-MS spectrum, the quasi-molecular ion peak was at m/z 227.06 [M-H] -. In the IR (KBr disk) spectrum showed absorption band at 2929 cm -1 and medium absorption band at 1466 cm -1 due to saturated C-H stretching. Medium absorption band at 3081 cm -1 and weak absorption band at 1640 cm -1 due to =C-H stretch.
The 13 C NMR spectrum revealed a total of fifteen carbon signals. Signals at δC138, 129.1, 128.95 and 126.5 attributed to mono substituted benzene ring, signals at δC128.6, 127, 126 and 115 due to alkene group in the structure. The signals at δC33.95 and 38.89 were assigned to aliphatic carbons and signals at δC43.1, 36 and 35.1 assigned for cyclic carbons. The multiplicity of each carbon atom was determined using DEPT-135 (table 3) Based on the 1D-NMR, IR , LC-MS, HMQC and HMBC spectra the proposed structure for the compound P2 was 1-(2-

Characterization of compound C3
Compound C3 was obtained as a yellow powdered substance from ethyl acetate extract and isolated from nhexane/EtOAc (7:3) ratio, the compound was characterized as follows. Its molecular formula, C16H30O3 was determined by negative LC-MS. In the negative LC-MS spectrum, the quasi-molecular ion peak was at m/z 271.02 In the IR (KBr disk) spectrum showed absorption strong band of the O-H stretch at 3434 cm -1 due to the presence of alcohol. Alkenes stretch at 3050 cm -1 . Strong absorption band at 2924 cm -1 and medium absorption band at 1465 cm -1 due to saturated C-H stretching. Strong absorption band at 1746 cm -1 due to ester group and absorption band at 1167 cm -1 due to C-O stretching. 1 H-NMR δH (400 MHz, DMSO-d6):-spectrum (table 3) revealed the presence of proton signals at δH 5.65(1H, d) and 5.54(1H, dt) attributed to alkenes protons. Signals at 3.54(3H) due to oxygenated methylene group and signals at δH1.00-2.2(20H, m) due to methylene protons. 13 C-NMR spectrum revealed a total of sixteen carbon signals. Signals at δC174 due to ester carbonyl group in the structure, signal at δC130 and 124 shows the presence of alkene. Signal at δC70.25 assigned for carbon bearing primary alcohol. The signals at δC34.19, 31.54, 29.47, 29.01, 27.9, 27.4, 24.98 and 22 were assigned to aliphatic carbons. The signal at δC27.5 was assigned to the carbons that bearing methyl substituent. Signal δC55.24 was assigned to the methyl carbon attached to oxygen. Signal at δC14.39 assigned for methyl carbon. The multiplicity of each carbon atom was determined using DEPT-135 experiment. Table 4 Based on the NMR (1D) and IR spectra (table 4) the tentative structure of compound C3 was proposed as (E)-methyl 14-hydroxy-10-methyltetradec-2-enoate. (Fig 3 below)

Conclusion
The study was undertaken to investigate phytochemical screening tests, isolation and characterization of compounds from seeds extract of Calpurnia aurea. As a part of our research, we have studied its chemical constituents and chemical structure elucidation of the isolated three compounds. The isolated compounds were identified as flavonoid compound G1 named: 2-(4-(2-hydroxyethyl)phenyl)-4H-chromen-4-one, P2 from methanol extract named: 1-(2-(3-((E)-buta-1,3-dienyl)cyclopentyl)ethyl)benzene and compound C3 from ethyl acetate extract named: (E)-methyl 14-hydroxy-10-methyltetradec-2-enoate. The chemical structures of the compounds were characterized on the basis of spectral data both 1D NMR and 2D NMR techniques were used to assign the NMR signals of the isolated compounds, including 1 H NMR, 13 C NMR, HMBC, HMQC, DEPT-135 and LC-MS spectra.