CHAPTER 8 ISOLATION AND CHARACTERIZATION OF PHYTOCONSTITUENTS BY COLUMN CHROMATOGRAPHY

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1 146 CHAPTER 8 ISLATIN AND CHARACTERIZATIN F PHYTCNSTITUENTS BY CLUMN CHRMATGRAPHY 8.1 INTRDUCTIN Column chromatography is an isolation technique in which the phytoconstituents are being eluted by adsorption. The principle involved in this separation of constituents is adsorption at the interface between solid and liquid. The component must have various degree of affinity towards adsorbent and also reversible interaction to achieve successful separation. No two compounds are alike in the above aspect. Low affinity compounds will elute first. The columns of different sizes were used for the present studies. Since the methanolic extract was found to possess significant pharmacological activity when compared to other extracts an attempt was made to fractionate the methanol extract by column chromatography. The elution was done by using solvents of different polarity like n- hexane, ethyl acetate and methanol. 8.2 MATERIALS AND METHDS Type of extract : Methanol extract Method : Dry packing method Packing material : Silica gel G

2 147 Procedure The methanol extract was subjected to Silica gel column chromatography for the isolation of the phytoconstituents. An appropriate column sized 5cm diameter and 50cm length was used. It was washed with water and rinsed with acetone and then dried completely. Little of pure cotton was placed at the bottom of column with the help of a big glass rod. Solvent hexane was poured into the column upto ¾ th level. Methanol extract was mixed with equal amount of graded silica gel until it became free flowing powder. When it reached a defined state it was slowly poured into the column containing hexane solvent with slight movement of stirring by glass rod to avoid clogging. Little cotton was placed on top of silica gel- extract mixture pack to get neat column pack. The knob at the bottom was slowly opened to release the solvent. The elution was done using hexane, ethyl acetate and methanol in different ratios like Hexane (100%- broad fraction 1), Hexane: Ethyl acetate (50:50- broad fraction 2), Ethyl acetate (100%- broad fraction 3), Ethyl acetate: Methanol (50:50 broad fraction 4) and methanol (100%- broad fraction 5). All the five broad fractions were collected separately and subjected to TLC. The solvents were evaporated by rotary vacuum evaporator. Since there was no yield in the Hexane fraction (100%) and very less yield in broad fractions 2 and 3, fractions 4, 5 were selected and again subjected to sub column fractionation.

3 148 Isolation of Compound I and II The 5 th fraction (methanol 100%) on concentrating on a rotary vacuum evaporator yielded light yellow sedimentation. It was then filtered and the light yellow powder was dried. The filtrate was concentrated and a brown semisolid was obtained. Both the fractions were subjected to thin layer chromatographic studies. The yellow coloured powder was named as compound I and the brown semisolid was named as compound II. Isolation of compound III The broad fraction 4 (Ethyl acetate: Methanol 50:50) was again subjected to sub column fractionation using different ratios of Iso propyl alcohol(ipa) and ethyl acetate. The fractions were collected and subjected to thin layer chromatographic studies. The similar fractions were mixed together and evaporated using rotary vacuum evaporator. The fractions (5% IPA and 95% Ethyl acetate) on evaporation yielded a light brown solid which was named as compound III. The pure compounds obtained were then subjected to spectral analysis for the determination of the structure of the compound. 8.3 TLC As soon as the fractions were eluted, it was analysed by using ready made TLC plate with suitable mobile solvent according to the polarity of elute. The developed chromatogram was observed under UV and also derivatized with detecting agent. 8.4 SPECTRAL ANALYSIS[ ] The isolated compounds were taken to determine the structure by instrumental spectral analysis such as

4 149 IR spectroscopy NMR - 1 H and 13 C IR Mass spectroscopy IR spectrum is considered as vibrational-rotational spectra. KBr pellet technique is used for solid compound, for liquid compound Nujol mull method is followed. It is very helpful record which would give information about functional group present in the organic compounds. Mechanism of bond stretching and bending is happened when electromagnetic radiation ranging from 500cm 1 to 4000 cm 1 passed through sample. Instrument used was ABD BWMEN Spectrometer NMR Nuclear magnetic spectrum is the most powerful spectral technique used to detect chemical structure of the molecules. The differences in the chemical environment around the different nuclei are exploited to obtain this information and is expressed in terms of chemical shifts in parts per million. When sample absorbs radiation at different radio frequency region which causes to excite type of proton or certain nuclei contained in the sample against magnetic field. The most commonly used NMR technique are the 1 H and 13 C. The instrument used was for detection is PRBHD Nuclear Magnetic Resonance spectrometer. 1 H NMR Most commonly used NMR is Proton NMR, because of the sensitivity and wide range of characteristic information. Range of chemical

5 150 shift ( ) from 0 14 ppm. Chemical shift of the test unknown compound was compared with TMS protons which are attributed at 0 ppm. But, the shift extends for the organic compound range 0 14 for the component. 13 C NMR It is new technique but natural abundance is very low 1.1%. So, this further reduces the sensitivity of the absorption. The range of chemical shift ) from ppm with use of TMS internal reference. An advantage is one can directly observe the functional group contained carbon atom Mass Spectroscopy It is an accurate method to determine the molecular mass of the compound. The main advantage is very small amount of sample is required for analysis than any other spectral methods. The mass spectroscopy uses the electric and magnetic fields to produce electrically charged ions of chemical substance under analysis. Instrument used to determine the spectrum was JEL Gcmate Mass spectrometer. The record spectrum tells about the mass, relative abundance of the molecular ions and positively charged fragments formed by electronic bombardment. Sample was dissolved in CDCl 3 and injected through direct probe inlet. Electronic impact ionization method was used.

6 RESULTS Column Chromatography Table 8.1 Broad fractions from methanol extract Eluent Solvent Ratio Compound n-hexane 100% - n-hexane: Ethyl acetate 50:50 - Ethyl acetate Ethyl acetate: Methanol 50:50 - Methanol 100 Compound-I Compound- II Table 8.2 Sub fractions from Ethyl acetate: Methanol(5:5) broad fraction Eluent Solvent Ratio Compound Ethyl acetate 100% - Ethyl acetate : IPA 95:5 Compound III Ethyl acetate : IPA 10:90 - Ethyl acetate: IPA 20:80 - Ethyl acetate: IPA 40: 60 - Ethyl acetate: IPA 60:40 - Ethyl acetate: IPA 80:20 - Ethyl acetate 100 -

7 152 SPECTRAL ANALYSIS The compounds (C-I, II and III) obtained with the methanol extract have identified and spectral data s were depicted in the Figures 8.1 to Characterization of Compound I Light yellow solid (1.5gm) was obtained which is soluble in methanol and water, the melting point was found to be C. The isolated compound answered the test for sugars. The various spectral data s obtained were given in Figures 8.1, 8.2, 8.3 and 8.4. Ir cm-1 : Nujol mull Wave Numbers (cm -1 ) Type CH 3, CH 2, CH 1695 C=C 1415 (C-) 1246 (C-) 1196 (C-) 1052 (C-) 897 (CH) 732 (CH)

8 153 Bruker NMR: 500MHz (solvent: D 2,,ppm), ppm: Parts per million Atom Carbon,, ppm Hydrogen,, ppm (d, 1H, J = 2.50 Hz) (d, 1H, J = 2.50 Hz) (t, 1H, J = 9.00 Hz) (t, 1H, J = 9.50 Hz) (m, 1H) Properties Name Glucuronic acid Molecular formula C 6 H 10 7 Molecular weight 194 Melting point C Mass (m/z) Colour Nature Solubility TLC studies 194[M+1] Yellow Solid Soluble in methanol and water Methanol: Ethyl acetate:water:acetic acid(2:6:1:1) Structure H 6 5 4

9 Figure 8.1 IR Spectrum of compound I 154

10 Figure H NMR Spectrum of compound I 155

11 Figure C NMR Spectrum of compound I 156

12 Figure 8.4 Mass spectrum of compound I 157

13 Characterization of Compound II Yellowish brown semisolid mass (2gm) was obtained which is soluble in methanol and water, the melting point was found to be C. The isolated compound answered the test for flavanoids. The various spectral data s obtained were given in the Figures 8.5, 8.6, 8.7 and 8.8. Ir cm-1 : Nujol mull WAVE NUMBERS (cm -1 ) TYPE CH 3, CH 2, CH 1686 C=C 1412 (C-) 1276 (C-) 1075 (C-) 913 (C-) 634 (CH) Bruker NMR (500MHz,,ppm in values):ppm:parts per million Atom Carbon,, ppm Hydrogen,, ppm (m, 1H) (m, 1H) (m, 1H) (m, 2H) (m, 1H) (m, 1H) (m, 1H) (m, 1H) (m, 1H) (m, 1H) (m, 1H) (m, 2H)

14 159 Properties Name Rhamnetin-3-- -D-Galactosyl D-galactopyranoside Molecular formula C 28 H Molecular weight 640 Melting point C Mass (m/z) 639( M ) + Colour Solubility TLC studies Nature Yellowish brown Soluble in methanol and water Methanol: Ethyl acetate:water:acetic acid(2:6:1:1) Semi-solid Structure H H H

15 Figure 8.5 IR Spectrum of compound II 160

16 Figure H NMR Spectrum of compound II 161

17 Figure C NMR Spectrum of compound II 162

18 Figure 8.8 Mass spectrum of compound II 163

19 Characterization of Compound III The compound III was obtained as brown solid and the melting point was found to be C. The isolated compound answered the test for triterpenoids. The various spectral data s obtained were given in the Figures 8.9, 8.10, 8.11 and Ir cm-1 : Nujol mull WAVE NUMBERS(cm -1 ) TYPE CH 3, CH 2, CH C= 1646 (C=C) 1367 (C-) 1076 (C-) 1035 (-CH) 824 (C-) 775 (C-H)

20 165 Bruker NMR (500MHz,, ppm in values) Atom Carbon,, ppm Hydrogen,, ppm

21 166 Atom Carbon,, ppm Hydrogen,, ppm Glycosides Linkages

22 167 Properties Name 3--[ -l-arabinopyranosyl-(1,4)-- -D- glucuronopyranosyl)]-31--( -D- glucopyranosyl) oleanolic acid Molecular formula C 48 H Molecular weight 932 Melting point C Mass (m/z) 825 ( M ) + Colour Nature Solubility Brown Sticky mass Soluble in water Structure ' H 17' 11' 10' 13' 14' 16' 15' 9' 7' ' H 1' 2' 3' 5' 4' 6'

23 Figure 8.9 IR Spectrum of compound III 168

24 Figure H NMR Spectrum of compound III 169

25 Figure C NMR Spetrum of compound III 170

26 Figure 8.12 Mass spectrum of compound III 171

27 DISCUSSIN Methanolic extract was found to possess significant pharmacological activity when compared to other extracts it was subjected to column chromatography for the isolation of phytoconstituents. Three compounds were isolated from the extract and their structures were identified by spectral studies. The structure of the isolated compound I was analysed by spectroscopic techniques like IR, NMR and mass spectroscopy. The melting point of the compound was found to be C and it answered the test for sugars. It had IR absorptions at 3391(hydroxyl), 2922 (C-H stretching of alkanes), 1686 (carbonyl group of acid), 1415, 1246, 1196 and 1052 (C- vibrations of alcohols). The 1 H NMR spectrum showed the presence of 5 protons on saturated carbon atoms. The value at 3.40 (d, 1H, J = 2.50Hz) and 3,92(d, 1H, J = 2.50Hz) showed the doublet protons. The other two protons were present as triplet with the value of 3.13(d, 1H, J = 9.00 Hz) and 3.48 (d, 1H, J = 2.50Hz). The value at 3.38 (M,1H) showed the presence of multiplet. The 13 C NMR spectrum showed peaks at 75.89, 95.85, 74.29, and corresponding to carbon connected to oxygen linkage. The peak at corresponds to the carbonyl carbon. Mass spectrum of the compound showed the molecular ion peak at m/z 194. From the spectral studies the possible structure of compound I may be glucoronic acid. The structure of the isolated compound II was analysed by spectroscopic techniques and the melting point of the compound was found to be C. It answered the test for flavanoids. It had IR absorptions at 3369(hydroxyl), 2930 (C-H stretching of alkanes), 1412,1276, 1075 and 913 (C- vibrations of alcohols) and 634 (C-H bending vibration of alkanes.

28 173 The 1 H NMR spectrum showed the presence of 14 protons on saturated carbon atoms. The protons are multiplet with the value of (M,H) in up field and the value at (M,H) showed the protons in the down field. The peak appeared in the range between ppm corresponds to carbon connected to oxygen linkage. 1 H and 13 C NMR spectrum of the compound showed the peaks at 73.41, 73.62, 75.25, 71.82, 72.40, 76.46, and indicated the carbon connected to oxygen linkage. Mass spectrum of the compound showed the molecular ion peak at m/z 639. From the spectral studies the possible structure of compound II may be Rhamnetin-3-- -D-Galactosyl D-galactopyranoside. The melting point of the compound III was found to be C and it answered the test for triterpenoids. It had IR absorptions at 3392(hydroxyl), 2926 (C-H stretching of alkanes), 1726 (C= stretching vibration of ketones), 1646 (C=C stretching vibration of alkanes), 1367, 1076 (C- stretching vibraton of alcohols), 1035 (C-H bending vibration of alkenes), 824(C- bending vibration of alcohols) and 725 (C-H bending vibration of alkanes). The 1 H NMR spectrum showed the presence of 50 protons in which 8 methyl protons were present with value of the 1H NMR signal at 3.20 was found to be secondary hydroxyl group. It showed the presence of olefinic proton with 5.16ppm. 13 C NMR spectrum showed peaks at 82.94, corresponds to carbon connected to oxygen. The peak at showed the presence of carbonyl carbon and at , showed the presence of double bond. Mass spectrum of the compound III showed the molecular ion peak at m/z 825. From the spectral studies the possible structure of compound II may be oleanolic acid derivative.