ether, Cholofrom, Ethyl acetate, Toluene, Formic Acid, Acetone, Methanol AR grade, S.D.
fine Chemicals, Ahmedabad), All the glass wares including round bottom flask, soxhlet
assembly, column, conical flask, volumetric flask, funnel, test tubes, pipettes, measuring
cylinder, beaker etc. were of Class A borosil glass, Calibrated micropipettes were used when
required for accurate measurement and transfer and Whatman no.41 filter paper, muslin cloth
was used as filtering and straining media.
Chemical Investigation for Separation and Isolation of Phytoconstituents
Extraction procedure
Air- dried powdered (100 gm) of O. basilicum seed sample were defatted by refluxing with
250 mL petroleum ether (60-80 °C) for 4 h. The materials were subjected to successive
solvent extraction using soxhlet apparatus with solvents in their ascending order of polarity
for 4 h and filtered through Whatman filter paper No 41. This procedure was repeated three
times to get complete extraction from the powder. All extracts were combined and evaporated
using vacuum-assisted rotary evaporator at 40 °C. The residue was weighed. All the dried
extract was stored in an air- tight container in refrigerator. From all extract extractive value of
methanolic extract is more. Methanolic extract shows the higher amount of phenolic and
flavonoids content. So dried methanolic extract used for further separation isolation, and
characterization of phytoconstituents from O. basilicum seed.
Separation of phytoconstituents using column chromatography
Column chromatography is one of the most useful method for the separation and purification
of plant constituents individually. In order to isolate the bioactive compound from the crude
extracts, they were further fractionated using column chromatography silica gel (40 g, 60-120
#, Spectrochem Pvt. Ltd.) as stationary phase. A cleaned and dried column (60 X 3cm) was
aligned in a vertical position. A clean and dried beaker was placed under the column outlet.
The column was partially filled with petroleum ether. A loose plug of cotton which had been
washed with petroleum ether was tamped down in to the bottom of the column. A small layer
of clean white sand was placed over the cotton wool by pouring sand in to the column. The
column was tapped gently to level the surface of the sand. The column was then filled with
slurry of silica gel prepared in hexane. The solvent was allowed to pass slowly from the
column. From the dried extract obtained as per section 6.1.3.1), 5 gm of extract was loaded on
a glass column (60 X 3 cm) packed with silica gel (40 g, 60-120#), as a stationary phase. The
extract was dissolved in a minimal quantity of methanol and adsorbed on to silica gel. When
the sample adsorbed to the silica gel, small amount of sand was poured to cover sample. The
mobile phase was poured continuously to the top of the column by aid of a funnel. The
61
Separation, Isolation and Characterization
bottom outlet of the column was opened. All the fractions were collected in separate test tube.
Colum was eluted with Toluene: Acetone: Formic acid system (5:4:1) until fraction were not
showing any spot on TLC. Total 50 fractions were collected and each fraction was checked by
TLC technique. Similar RF value containing fractions were mixed together, allowed to
evaporate to remove solvent. These fractions were grouped together according to their
homogeneity, judged from the TLC analysis.
Purification of crystals
Dried residue of similar RF value containing fractions was suspended in 50 mL of ethyl
acetate. The extraction procedure with ethyl acetate was carried out three times. Combined
extract was evaporated to its 1/4 volume (30 mL) on an electric water bath and semi solid
mass obtained which keep aside for 5- 6 h which developed crystals. Recrystallization is
carried out to obtain pure crystals using methanol and pure crystal assigned as compound OB-
I, OB-II and OB-III.
Identification and characterization of the isolated phytoconstituent using M.P, UV, FT-
IR, Mass, NMR spectroscopy
Pure crystals obtained as per section 6.1.3.3 is designated as OB-I, OB-II and OB-III. The
structure of a purified compound can be determined using information from various
spectroscopic techniques. The isolated and purified crystals of OB-I, OB-II and OB-III was
analysed by melting point and different spectroscopic techniques like UV, FT-IR, Mass, 1H
and 13C NMR spectroscopy for characterization and structural elucidation.
Melting point
Melting point of all crystals was checked using melting point apparatus and compared with
literature value.
UV spectroscopy
To determine absorbance maxima, methanolic solution of OB-I, OB-II and OB-III prepared
and was scan in UV-Visble region from 200-800 nm.
FT-IR spectroscopy
The FT-IR spectra of OB-I, OB-II and OB-III was recorded in the range of wave number 400
- 4000 cm-1.
Mass spectroscopy
The analysis was performed in positive ionization mode with Electrospray interface. The mass
to charge (m/z) ratio was recorded in the range of 50-500 m/z. The parameters for capillary
and Radio frequency voltage were 80 V, with nebulizer gas as air at a pressure of 35 psi and
curtain gas as nitrogen at a pressure of 10 psi.
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Chapter 6
NMR spectroscopy
OB-I, OB-II and OB-III (50 mg) of each has been taken and dissolved in DMSO. 1H NMR
and 13C NMR spectra of OB-I, OB-II and OB-III were recorded on instrument as described in
section 6.1.
RESULTS AND DISCUSSION
Separation and isolation of phytoconstituents from methanolic extract of O. basilicum seed
was carried out using column chromatographic method.
Optimization of mobile phase
Thin layer chromatography was used for the separation of phytoconstituents. Methanolic
extract was applied to a commercially prepared TLC (Silica Gel 60F254) plate with different
solvent systems i.e. the aim of this procedure was to identify the number of components
present in the extract, separation between components which can further helpful optimization
column chromatography. Different solvent system was tried for optimization of mobile phase
like Butanol: Acetic acid : Water (14:1:5), CHCl3: Ethyl acetate (6:4), Toluene: Ethyl acetate:
Formic acid (6:6:1), Toluene: Ethyl Acetate (9.5:0.5), Toluene: Acetone: Formic acid (5:4:1)
etc. (Ramasubramania, Sathyanathan, and Sekhar, V. and Roosewelt).
Visualization was done under UV light and spraying with concentrated Fast blue B salt MS
reagent, drying in the oven at 105 ºC. The spots were visualized, labelled and their
retardation factors (RF value) were calculated and compared. The RF values were calculated
according to the following formula:
RF = Distance from starting point to center of spot
Distance from starting point to solvent front
Only one spot was observed in Butanol: Acetic acid, Water (14: 1: 5, v/v) and Toluene:
Ethyacetate (9.5: 0.5, v/v), Two spots were observed Toluene: Acetone: Formic acid (6: 6: 1,
v/v). Optimization of the chromatographic conditions were achieved with mobile phase
containing toluene: acetone: formic acid (5: 4: 1, v/v/v) at RF Values 0.72. 0.65 and 0.28.
63
Separation, Isolation and Characterization
Table 6.1 Thin layer chromatography for methanolic extract of O. basilicum seed
RF Values
Sr. No. of
Mobile Phase Composition After
No. spots 254 nm 366 nm
Derivatization
Butanol: Acetic acid : Water
1 1 - - 0.5
(14: 1: 5, v/v)
Toluene: Ethyl Acetate
2 1 - 0.5 0.6
(9.5: 0.5, v/v)
Toluene: Acetone: Formic 0.23,
3 2 - 0.25, 0.67
acid (6: 6: 1, v/v) 0.65
0.72,
Toluene: Acetone: Formic 0.72, 0.65,
4 3 0.65, -
acid (5: 4: 1, v/v) 0.28
0.28
Figure 6.1 TLC plate of methanolic extract of O. basilicum seed showing spots at
different RF at 254 nm
64
Chapter 6
Figure 6.2 TLC plates of methanolic extract of O. basilicum seed showing spots at 366
nm after spraying with Fast blue B salt MS
Separation of phytoconstituents by column chromatographic method
Total fifty fractions were collected and each fraction was checked by TLC technique. Similar
RF value containing fractions were mixed together, allowed to evaporate to remove the
solvent. These fractions were grouped together according to their homogeneity, judged from
the TLC analysis. Among all the fractions collected, the fractions from 9-12, 22-27 and 44-49
gave a three different compound with RF value of 0.72, 0.65 and 0.28 respectively and
assigned as compound OB- I, OB-II and OB-III. TLC profile of isolated compound by
column chromatography is as shown in Table 6.2.
Table 6.2 TLC profile of isolated compound by column chromatography
Test No. of
Sr. No RF Values % yield
Tube No. Spots
1 9-12 1 0.72 0.2
2 22-27 1 0.65 0.16
3 44-49 1 0.28 0.14
65
Separation, Isolation and Characterization
66
Chapter 6
Theoretical wave numbers responsible for functional groups are compared with observed
wave numbers and presented in Table 6.5. IR spectra of isolated compound OB-I and standard
apigenin as shown in Figure 6.4 and 6.5 respectively.
67
Separation, Isolation and Characterization
68
Chapter 6
Figure 6.8 - 6.12. H 1 NMR (400 MHz, DMSO-d6): δ 12.91 (s, 1 H, 5-OH) , 10.43 (s, 2 H,
7-OH/4’-OH), 7.85 (dd, 2 H, H-2’, 6’), 6.94 (dd, 2 H, H-3’, 5’) , 6.64 (s, 1 H, H-3) , 6.43
(d, 1 H, H-8) 13C NMR (100 MHz, DMSO-d6): δ 181.59 (C-4), 163.99 (C-7) 163.62 (C-2),
161.48 (C-5), 161.20 (C-4’), 157.24 (C-9), 128.03 (C-2’, 6’) 121.19 (C-3’, 5’), 115.81(C-
1’),103.73 (C-10), 102.72 (C-3) 98.76 (C-6), 93.75 (C-8).
69
Separation, Isolation and Characterization
70
Chapter 6
71
Separation, Isolation and Characterization
72
Chapter 6
1
H NMR(δ) (400 MHz, DMSO-d6) 500 MHz, DMSO-d6)
δ:12.91 (s, 1 H, 5-OH) δ:12.97 (1H, s, 5-OH)
10.43 (s, 2 H, 7-OH/4’-OH) 10.82 (1H, s, 7-OH),
7.85 (dd, 2 H, H-2’, 6’) 10.35 (1H, s, 4′-OH),
6.94 (dd, 2 H, H-3’, 5’) 7.93 (2H, d, H-2′ ,6′),
6.64 (s, 1 H, H-3) 6.93 (2H, d, H-3′, 5′),
6.43 (d, 1 H, H-8) 6.79 (1H, s, H-3),
6.19 (d, 1 H, H-6) 6.48 (1H, d, H-8),
6.20 (1H, d, H-6),
13 13 13
C C NMR (100 MHz, DMSO-d6) C-NMR (125 MHz,
NMR(δ) δ:181.59 (C-4), 163.99 (C-7) DMSO-d6)
163.62 (C-2), 161.48 (C-5), δ:181.75 (C-4), 164.29 (C-7),
161.20 (C-4’), 157.24 (C-9), 163.72 (C-2), 161.46 (C-5),
128.03 (C-2’, 6’) 121.19 (C-3’, 161.20 (C-4’), 157.33 (C-9), (Liu et al.)
5’), 115.81(C-1’),103.73 (C-10), 128.48 (C-2’, 6’), 121.17 (C-
102.72 (C-3) 98.76 (C-6), 93.75 3’, 5’),115.97 (C-1’), ,
(C-8) 103.65 (C-10), 102.82(C-3),
98.87 (C-6), 93.99 (C-8
MASS [M+H]+ 271.38 (100) [M+H]+ at 271.58 (100)
Based on the results of spectral data of OB-I, the compound may be apigenin, which was
further confirmed by comparing spectral data in the literature. Figure 6.13 shows the structure
of apigenin.
Introduction to apigenin
Apigenin (4, 5, 7-trihydroxyflavone) is a natural product which belongs to the flavones class
which is a subclass of flavonoid that is the aglycone part of several naturally occurring
glycosides. From which, common constituents are plant deprived, fruits, vegetables, sprouts,
73
Separation, Isolation and Characterization
wheat and some seasonings. Chamomile tea, grapefruits, onions, oranges, and some spices
like parsley are among the important and popular sources. It is an active ingredient of Gingko
biloba, a memory herb and found in higher levels in yarrow, liquorice, cilantro, foxglove,
horehound, red wine, spearmint, flax seed, passion flower, coneflower, celery, tarragon
(Sanjeev Shukla).
Physicochemical properties of apigenin
Molecular weight: 270.24 gm/mole
Molecular formula: C15H10O5
Colour: Yellow crystalline powder
Solubility: It is practically insoluble in water, moderately soluble in hot alcohol and soluble in
dilute KOH, DMSO (S. Budavari)
Pharmacological properties
Anti-inflammatory, antiviral, antibacterial, antioxidant, vasodilatory, hepatoprotective,
locomotor, also its role as a chemo preventive agent in cancer in an organ-specificity format like
breast, colon, lung, prostate, ovarian, thyroid, skin, endocrine, gastric, adrenal gland, pancreas
and liver. (Parikh and Kothari).
6.2.3.7. 3 Storage Condition
Store in -20 °C. Keep away from direct sunlight (S. Budavari).
Identification and Characterization of Isolated Compound OB-II Using Melting point,
UV, FT-IR, Mass, NMR spectroscopy
Checking melting point of OB-II Melting
point of OB-II is shown in Table 6.7 Table 6.7
Melting point of compound OB- II
Literature value
Compound Observed Melting point
(Hye et al.)
OB-II 176- 178 °C 175-177 °C
Results of UV spectroscopy
UV spectrum of OB-II recorded in methanol in the range of 200-400 nm on UV
spectrophotometer. UV spectra of the compound show maximum absorbance at 279 nm as
shown in Figure 6.14 and value of λ max reported in Table 6.8
74
Chapter 6
75
Separation, Isolation and Characterization
76
Chapter 6
291.2331
3908
107.0690
1015
243.0815 431.4581
503 522
0 m/z
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000
291.2331
3908
107.0690
1015
243.0815 272.2246 431.4581
503 497 522
0 m/z
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520
77
Separation, Isolation and Characterization
78
Chapter 6
79
Separation, Isolation and Characterization
80
Chapter 6
81
Separation, Isolation and Characterization
4.89 (d, 1H, H-2), 5.70(d, H-8), 5.89 (1H, H-6), 6.59
1H, H-5), 5.90 (d, 1H, H- (1H, H-6′), 6.68 (1H, H-5′),
8), 6.59 (m, 1H, H-6′), 6.72 (1H, H-2′), 9.21 (S, 1H,
6.70 (m, 1H, H-5′), 6.73 (1H, H-3’), 8.97 (S, 1H, H-7) 8.90 Hye et al.
H-2′), 9.21 (S, 1H, H-3’), (S, 1H, H-4’), 8.85 (S, 1H, H-
8.97 (S, 1H, H-7) 8.90 (S, 6)
1H, H-4’), 8.85 (S, 1H, H-6)
13
C NMR(δ) 100 MHz, DMSO 500 MHz, DMSO
27.8 (C-4), 66.2 (C-3), 27.8 (C-4), 66.3 (C-3),
80.9 (C-2), 93.78 (C-8), 81.0 (C-1), 93.8 (C-8),
95.03 (C-6), 98.9 (C-4a), 95.1 (C-6), 99.0 (C-4a),
114.4 (C-2′), 115.0 (C-5′), 114.5 (C-2′), 115.0 (C-5′),
118.4 (C-6′), 130.5 (C-1′), 118.4 (C-6′), 130.6 (C-1′),
144.7 (C-3′, 4′), 155.3 (C-8a), 144.8 (C-3′, 4′), 155.3 (C-8a),
156.1 (C-5), 156.4 (C-7). 156.1 (C-5), 156.4 (C-7).
MASS m/z: 290 [M+]+ m/z: 291 [M+H]+
Based on the results of spectral data of OB-II, the compound may be catechin, which was
further confirmed by comparing spectral data in literature. Figure 6.25 shows the structure of
catechin.
Introduction to catechin
Catechin is flavanol, which is also called proanthocyanidins or flavan-3-ols. Catechin is
naturally present in fruits, vegetables, tea and wine, green, black and oolong teas, fruits like
plum, apples, peach, strawberry and cherry, and beans and grains like broad bean, lentil and
cocoa. (Yilmaz).
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Chapter 6
Results of UV spectroscopy
UV spectrum of OB-III recorded in methanol in the range of 200-400 nm on UV
spectrophotometer. UV spectra of compound show maximum absorbance at 271 nm as shown
in Figure 6.26 and value of λ max reported in Table 6.12.
83
Separation, Isolation and Characterization
84
Chapter 6
85
Separation, Isolation and Characterization
86
Chapter 6
87
Separation, Isolation and Characterization
88
Chapter 6
Based on the results of spectral data of OB-III, the compound may be Gallic acid, which was
further confirmed by comparing spectral data in the literature. Figure 6.32 shows the structure
of Gallic acid.
89
Separation, Isolation and Characterization
90
Chapter 6
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Parikh, Nisha H, and Charmy S Kothari. “Development and Validation of a High-
Performance Thin-Layer Chromatographic – Densitometric Method for the
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29.3 (2016): 216–220.
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