Separation, Isolation and Characterization

6 SEPARATION, ISOLATION AND CHARACTERIZATION OF

PHYTOCONSTITUENT
Extraction is the first crucial step in the analysis of medicinal plants, because it is necessary to
extract the desired chemical components from the plant materials. It is required for further
separation and characterization (Sasidharan S, Y Chen). In the plant material, secondary
metabolites usually are found inside cells. Thus grinding of the raw material and breaking
tissue and cell integrity increases extraction yield (Franz Bucar a). Based on the literature
survey, it was observed that different extracts of seed showed potent therapeutic effect.
Preliminary phytochemical screening for the seed of O. basilicum revealed the presence of
flavonoids, carbohydrate, glycosides, phenolic etc. These phytoconstituents are required to be
separated and isolated. As the target compounds may be non-polar to polar, thermally labile,
the suitable method for the extraction must be selected. Various methods, such as sonication,
heating under reflux, soxhlet extraction are commonly used for the plant extraction samples.
In the present chapter, seed of O. basilicum is used for the preparation of extract and used for
further separation. Among all, methanolic extract gave (5.0 gm) yield which was higher
compared to all which also contain higher amount of phenolic and flavonoid content. Based
on phytochemical screening, methanolic extract was further chosen for separation and
isolation of compounds by column chromatography. Pure crystals obtained by were
characterized using various spectroscopic techniques like UV, IR, NMR, Mass spectroscopy.
EXPERIMENTAL
Instruments and Equipments
Sihmadzu Aux 120 (Gottingen, Germany) analytical., UV (A Camag ) cabinet with dual
wavelength UV lamp (254 nm and 366 nm).,Water bath, Janki Impex, Pvt. Ltd., Ahmedabad,
India, A UV-Visible spectrophotometer (UV1800; Shimadzu, Kyoto, Japan) with wavelength
accuracy of ± 0.5 nm, a pair of 10 mm matched quartz cells with 2 mm spectral width, Rotary
Evaporator model RE301 (MRS scientific, UK)., Melting point apparatus, H305/0162, Janki
Impex, Pvt. Ltd., Ahmedabad, India., Fourier Transform Infrared Spectroscope, (FT/IR-
4600) Jasco, Japan., Water, Q-TOF Micro mass spectrometer,New Delhi., Bruker advance II
NMR spectrometers (Bruker Ltd. Switzerland).
Chemicals, Reagents and Materials
Apigenin, Catechin, and Gallic Acid (>98% pure) were procured from (Sigma-Aldrich
(India), Fast blue B salt MS (S.D. fine Chemicals, Mumbai (India), Silica Gel 60-120#
Spectrochem Pvt. Ltd, Mumbai, India, Pre-coated with silica gel 60 F254 with( 0.2 mm)
layer thickness Aluminum sheet for HPTLC plates (20 x 20 cm, Merck, Germany), Petroleum
60
Chapter 6

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.

62
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.

Chemical Investigation for Separation Isolation and of Phytoconstituents

TLC investigation
Separation of compounds has been achieved by using different solvent systems as shown in
Table 6.1. Remarkable separation of components present in methanolic extract of O.
basilicum seed are achieved by toluene: acetone: formic acid (5:4:1) as shown in Figure 6.1
and 6.2

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

Identification and Characterization of Isolated Compound OB-I using Melting point,

UV, FT-IR, Mass, NMR spectroscopy
Checking melting point of OB-I Melting
point of OB-I is shown in Table 6.3. Table
6.3 Melting point of compound OB- I
Reported value
Compound Observed Melting point
(Liu et al.)
OB-I 346 -348 °C 345-350 °C

65
 Separation, Isolation and Characterization

6.2.2.2 Results of UV spectroscopy

UV spectrum of OB-I recorded in methanol in the range of 200-400 nm on UV
spectrophotometer. UV spectra of the compound shows maximum absorbance at 340 nm as
shown in Figure 6.3 and value of λmax reported in Table 6.4.

Figure 6.3 UV spectra of compound OB-I in Methanol (200-400 nm)

Table 6.4 λmax of compound OB- I

Compound Observed λmax Reported λmax
(Liu et al.)
OB-I 340 nm 340 nm

6.2.3.3 Results of FT-IR spectroscopy

FT-IR spectroscopy was carried out to ascertain functional groups. FT-IR spectrum of OB-I
as shown in Figure 6.5 was recorded in diffused reflectance mode.
The FT-IR spectrum of OB-I presents dominant IR absorption bands in the high wave region
at 3319 cm−1, and 2923 cm−1 for -OH, and -CH3 asymmetric and symmetric stretching
vibrations, respectively.
In finger print region, the FT-IR spectrum presents dominant bands at 1650, 1456, 1352,
1116, 827 cm-1 and many other bands of medium to weak intensity. The observed band at
1650 cm-1 can be assigned to C=O stretching. Other bands in the spectral range are assigned
to bending vibrations of -OH, and -CH3 groups as well as to skeletal bending bonds. The
intense band as 827 cm-1 in IR spectrum is due to vibration of the -CH2 in alkene group.

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.

Figure 6.4 Recorded FT-IR spectra of compound OB-I

Figure 6.5 Recorded FT-IR spectra of standard Apigenin

67
 Separation, Isolation and Characterization

Table 6.5 Important frequencies of OB-I obtained in FT-IR spectra

Observed Reported
Compound
Functional group frequency frequency (cm-1)
Parameter
(cm-1) (Nayaka et al.)
-OH stretching 3319 3290
-OH Bending 1456 1497
IR (cm-1) -C=O stretch of -C=O group 1650 1655
-C-O stretching of -C=O
1116 1114
group

6.2.2.4 Results of Mass spectroscopy

Mass spectroscopy (MS) was carried out to determine the molecular weight of the isolated
phytoconstituent. The MS and MS/MS spectra of OB-I are shown in Figure 6.6 and 6.7. The
molecular ion [M+H]+ peak was obtained at 271.38 m/z which conforms molecular weight of
OB-I at 270.24.

Figure 6.6 Mass spectra of OB-I

68
Chapter 6

Figure 6.7 Mass spectra of OB-I

Results of NMR spectroscopy
The H and 13C NMR experiments of OB-I were carried out. Results are mentioned in
1

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

Figure 6.8 1H NMR spectra of OB-I

Figure 6.9 1H NMR spectra of OB-I

70
Chapter 6

Figure 6.10 13C NMR spectra of OB-I

Figure 6.11 13C NMR spectra of OB-I

71
 Separation, Isolation and Characterization

Figure 6.12 13C NMR spectra of OB-I

Summarized results of spectral analysis
Results obtained from of M.P., IR, Mass and NMR spectra of the isolated phytoconstituent as
shown in Figure 6.4 - 6.12, it was proposed that the structure may be Apigenin; observed data
are compared with reported data as shown in table 6.6 which confirms the structure of
apigenin. Figure 6.13 shows the structure of Apigenin.
Table 6.6 Summarized results of spectral analysis of OB-I
Parameter OB-I Literature value Reference
(Reported value)
M.P. 346-348 °C 345-348 °C (Liu et al.)
UV (λmax) 340 nm 340 nm (Liu et al.)
IR (cm-1) -OH (3319 cm-1) -OH (3310 cm-1) (Nayaka et
-CH (1456 cm-1) -CH (1451 cm−1 ) al.)
-C=O (1650 cm-1) -C=O (1647 cm−1)
-C−O (1116 cm-1) -C−O (1114 cm−1)

72
Chapter 6

1
H NMR(δ) (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)
6.19 (d, 1 H, H-6)
13 13
C C NMR (100 MHz, DMSO-d6)
NMR(δ) 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)
MASS [M+H]+ 271.38 (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.
500 MHz, DMSO-d6)
δ:12.97 (1H, s, 5-OH)
10.82 (1H, s, 7-OH),
10.35 (1H, s, 4′-OH),
7.93 (2H, d, H-2′ ,6′),
6.93 (2H, d, H-3′, 5′),
6.79 (1H, s, H-3),
6.48 (1H, d, H-8),
6.20 (1H, d, H-6),
13
C-NMR (125 MHz,
DMSO-d6)
δ:181.75 (C-4), 164.29 (C-7),
163.72 (C-2), 161.46 (C-5),
161.20 (C-4’), 157.33 (C-9), (Liu et al.)
128.48 (C-2’, 6’), 121.17 (C-
3’, 5’),115.97 (C-1’), ,
103.65 (C-10), 102.82(C-3),
98.87 (C-6), 93.99 (C-8
[M+H]+ at 271.58 (100)

Figure 6.13 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

Figure 6.14 UV spectra of compound OB-II in Methanol

Table 6.8 λmax of compound OB- II

Compound Observed λmax Reported λmax (Hye et al.)
OB-II 279 nm 279 nm

Results of FT-IR spectroscopy

The FT-IR spectrum of OB-II presents dominant IR absorption bands in the high wave region
at 3392 cm−1 , 2916 cm−1 and 2825 cm−1 for –OH, -CH3 and -CH2 asymmetric and symmetric
stretching vibrations respectively.
In finger print region, the FT-IR spectrum presets dominant bands at 1681, 1425, 1352, 1107,
885 cm-1 and many other bands of medium to weak intensity. The observed band at 1654 cm -1
can be assigned to C=O stretching of - COOH functional group. Other bands in the spectral
range are assigned to bending vibrations of –OH, - CH2 and CH3 groups as well as to skeletal
bending bonds. The intense band as 885 cm -1 in IR spectrum is due to vibration of the CH2 in
alkene group. Theoretical wave numbers responsible for functional groups are compared with
observed wave numbers and presented in Table 6.9. IR spectra of isolated compound OB-II
and standard catechin as shown in Figure 6.15 and 6.16 respectively.

75
 Separation, Isolation and Characterization

Table 6.9 Important frequencies of OB-II obtained in FT-IR spectra

Theoretical
Compound Observed
Functional group frequency (cm-1)
Parameter frequency (cm-1)
(Hye et al)
-OH stretching of carboxylic
3392 3231
group
-OH Bending 1450 1468
-C=O stretch of -C=O group 1681 1624
IR (cm-1)
-C-O stretching of
1107 1111
-C=Ogroup

Figure 6.15 IR spectra of compound OB-II

Figure 6.16 IR spectra of standard catechin

76
Chapter 6

Results of Mass spectroscopy

The MS and MS/MS spectra of OB-II are shown in Figure 6.17 and 6.18. The molecular ion
[M+] peak was obtained at 290.18 m/z which confirms molecular weight of OB-II at 290.27

WATERS, Q-TOF MICROMASS (ESI-MS) SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH

NISHA E-1 8 (0.149) Cm (5:14-23:28) TOF MS ES+
290.1953 1.42e4
100 14153
%

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

Figure 6.17 Mass spectra of compound OB-II

WATERS, Q-TOF MICROMASS (ESI-MS) SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
NISHA E-1 8 (0.149) Cm (5:14-23:28) TOF MS ES+
290.1953 1.42e4
100 14153
%

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

Figure 6.18 Mass spectra of compound OB-II

77
 Separation, Isolation and Characterization

Results of NMR spectroscopy

The 1H and 13
C NMR experiments of OB-II were carried out results obtained mentioned in
Figure 6.19 - 6.24. The 1H and 13C NMR experiments of OB-II were carried out. Results are
mentioned in Figure 6.19 - 6.24. 1
H-NMR (DMSO-d6) δ: 2.35 (dd, 1H, H-4), 2.67 (dd,
1H, H-4), 3.84 (m, 1H, H-3), 4.50 (d, 1H, H-3), 4.90 (d, 1H, H-2), 5.70(d, 1H, H-5), 5.90 (d,
1H, H8 6.59 (m, 1H, H-6′), 6.70 (m, 1H, H-5′), 6.73 (1H, H-2′), 9.21 (S, 1H, H-3’), 8.97 (S,
1H, H-7) 8.90 (S, 1H, H-4’), 8.85 (S, 1H, H-6) .13C NMR(δ) 100 MHz, DMSO: 27.8 (C-4),
66.2 (C-3), 80.9 (C-2), 93.78 (C-8), 95.03 (C-6), 98.9 (C-4a), 114.4 (C-2′), 115.0 (C-5′), 118.4
(C-6′), 130.5 (C-1′), 144.7 (C-3′, 4′), 155.3 (C-8a), 156.1 (C-5), 156.4 (C-7).

Figure 6.19 1H NMR spectra of OB-II

78
Chapter 6

Figure 6.20 1H NMR spectra of OB-II

Figure 6.21 1H NMR spectra of OB-II

79
 Separation, Isolation and Characterization

Figure 6.22 1H NMR spectra of OB-II

Figure 6.23 1H NMR spectra of OB-II

80
Chapter 6

Figure 6.24 13 C NMR spectra of OB-II

Summarized results of spectral analysis
Results obtained from of M.P., UV, IR, Mass and NMR spectra of the isolated
phytoconstituent as shown in, Figure 6.16 - 6.24, it was proposed that the structure may be
catechin, observed data are compared with reported data as shown in table 6.10 which
confirms the structure of catechin. Figure 6.25 shows the structure of catechin.
Summarized results of spectral analysis of OB-II are as shown in Table 6.10.
Table 6.10 Summarized results of spectral analysis of OB-II
Parameter OB-II Literature value Reference
(Reported value)
M.P. 176 -178 °C 175-177 °C
UV (λ max) 278 nm 278 nm
IR (cm-1) 3321,3392,1650,1515, 1471, 2600-3400 (broad), 1620,
1384,1240, 1164, 1107, 1080, 1520, 1470, 1380, 1240, 1150,
1026, 835 cm-1. 1120, 1080, 1020, 820 cm-1.
1
H NMR(δ) 400 MHz, DMSO 400 MHz, DMSO
δ: 2.35 (dd, 1H, H-4), δ: 2.35 (1H, dd, H-4), 2.66
2.67 (dd, 1H, H-4), 3.82 (m, (1H, dd,, H-4), 3.81 (1H, H-3),
1H, H-3), 4.50 (d, 1H, H-3), 4.48 (1H, d, H-2), 5.69 (1H,

81
 Separation, Isolation and Characterization

4.89 (d, 1H, H-2), 5.70(d,
1H, H-5), 5.90 (d, 1H, H-
8), 6.59 (m, 1H, H-6′),
6.70 (m, 1H, H-5′), 6.73 (1H,
H-2′), 9.21 (S, 1H, H-3’),
8.97 (S, 1H, H-7) 8.90 (S,
1H, H-4’), 8.85 (S, 1H, H-6)
H-8), 5.89 (1H, H-6), 6.59
(1H, H-6′), 6.68 (1H, H-5′),
6.72 (1H, H-2′), 9.21 (S, 1H,
H-3’), 8.97 (S, 1H, H-7) 8.90 Hye et al.
(S, 1H, H-4’), 8.85 (S, 1H, H-
6)

13
C NMR(δ) 100 MHz, DMSO
27.8 (C-4), 66.2 (C-3),
80.9 (C-2), 93.78 (C-8),
95.03 (C-6), 98.9 (C-4a),
114.4 (C-2′), 115.0 (C-5′),
118.4 (C-6′), 130.5 (C-1′),
144.7 (C-3′, 4′), 155.3 (C-8a),
156.1 (C-5), 156.4 (C-7).
MASS m/z: 290 [M+]+
500 MHz, DMSO
27.8 (C-4), 66.3 (C-3),
81.0 (C-1), 93.8 (C-8),
95.1 (C-6), 99.0 (C-4a),
114.5 (C-2′), 115.0 (C-5′),
118.4 (C-6′), 130.6 (C-1′),
144.8 (C-3′, 4′), 155.3 (C-8a),
156.1 (C-5), 156.4 (C-7).
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.

Figure 6.25 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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Physicochemical properties of catechin

Molecular weight: 290.27gm/mole
Molecular formula: C15H14O6
Colour: Colourless solid.
Solubility: It is soluble in ethanol, methanol, DMSO and dimethyl formamide.
Optical rotation: +14.0°(“Catechin | The Merck Index Online”)
Pharmacological properties
Catechin shows anti-oxidant, anti-cancer, anti ulcer, hepatoprotective, anti-obesity, anti
arthritic, anti-inflammatory, anti-immunostimulant, anti hyperlipidemic, antispasmodic,
bronchodilator and vasodilator activities (Shashank and Abhay)(Ghayur MN, Khan H).
6.2.4.7.3. Storage condition
It is recommended to store at low temperature (4 °C) (“Catechin | The Merck Index Online”).
Identification and Characterization of Isolated Compound OB-III Using Melting point,
UV, FT-IR, Mass, NMR spectroscopy
Checking melting point of OB-III Melting
point of OB-III is shown in Table 6.11 Table
6.11 Melting point of compound OB- III
Compound Observed Melting point Reported value
(Ashford)
OB-III 259 -261 °C 260-261 °C

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.

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 Separation, Isolation and Characterization

Figure 6.26 UV spectra of compound OB-III

Table 6.12 λ max of compound OB- III

Reported λ max
Compound Observed λ max
(Abd El Azim)
OB–III 271 nm 271 nm

Results of FT-IR spectroscopy

The FT-IR spectrum of OB-III presents dominant IR absorption bands in the high wave
region at 3462 cm−1, 2997 cm−1 and 2705 cm−1 for –OH, -CH3 and -CH2 asymmetric and
symmetric stretching vibrations respectively.
In finger print region, the FT-IR spectrum presets dominant bands at 1693, 1467, 1373, 1101,
866 cm-1 and many other bands of medium to weak intensity. The observed band at 1693 cm -1
can be assigned to -C=O stretching of -COOH functional group. Other bands in the spectral
range are assigned to bending vibrations of -OH, -CH2 and -CH3 groups as well as to skeletal
bending bonds. The intense band as 866 cm-1 in IR spectrum is due to vibration of the CH2 in
alkene group. Theoretical wave numbers responsible for functional groups are compared with
observed wave numbers and presented in Table 6.13. IR spectra of isolated compound OB-II
and standard Catechin as shown in Figure 6.27 and 6.28 respectively.

Table 6.13 Important frequencies of OB-III obtained in FT-IR spectra

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Compound Functional group Observed Reported

Parameter frequency (cm-1) frequency (cm-1)
(Abd El azim)
-OH stretching of carboxylic 3462 3491
group
IR (cm-1) -OH Bending 1467 1453
-C=O stretch of –C=O group 1681 1703
-C-O stretching of –C=Ogroup 1107 1101

Figure 6.27 IR Spectra of compound OB-III

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 Separation, Isolation and Characterization

Figure 6.28 IR Spectra of compound standard catechin

Results of Mass spectroscopy

The MS spectra of OB-III are shown in Figure 6.29. The molecular ion [M+] + peak was
obtained at 170.0 m/z which confirms molecular weight of OB-III at 170.11.

Figure 6.29 Mass Spectra of compound OB-III

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Results of NMR spectroscopy

The 1H and 13
C NMR experiments of OB-III were carried out results obtained mentioned in
Figure 6.30- 6.31. 1H-NMR 400 MHz, (DMSO- d6) 6.98 (s, 2H, H-2 and H-6). 13
C NMR
(DMSO) 167.45 (C-1), 145.67 (C-3 and C-5), 137.93 (C-4), 120.37 (C-2), 108.66 (C-3 and
C7).

Figure 6.30 1 H NMR Spectra of compound OB-III

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 Separation, Isolation and Characterization

Figure 6.31 13 C NMR Spectra of compound OB-III

Summarized results of spectral analysis

Results obtained from of M.P., UV, IR, Mass and NMR spectra of the isolated
phytoconstituent, as shown in Figure 6.26- 6.31, it was proposed that the structure may be of
Gallic acid, observed data are compared with reported data as shown in table 6.14 which
confirms the structure of Gallic acid. Figure 6.32 shows the structure of Gallic acid.

Table 6.14 Summarized results of spectral analysis of OB-III

Literature value
Parameter OB-III Reference
(Reported value)

M.P. 258-261 °C 260 °C (Ashford)

UV (λ max) 271 nm 271 nm (Abd El Azim)

IR (cm-1) 3461,3328,1693,1539, 3491, 3377, 1703, (Abd El Azim)

1467, 1373, 1238, 1617, 1539, 1453,

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Chapter 6

1164, 852 cm-1. 1254

1
H NMR(δ) 400 MHz, DMSO- d6 400 MHz, DMSO-d6 (Abd El Azim)
6.98(s, 2H, H-2 and 6.98(s, 2H, H-2 and
H-6). H-6).
13
C NMR(δ) 167.45 (C-1), 167.7(C-1). (Abd El Azim)
145.67 (C-3 and C-5), 145.5(C-3 and C-5),
137.93 (C-4), 138.1(C- 4),
120.37 (C-2), 120.6(C-2),
108.66 (C-3 and C7) 108.8(C-3 and C-6),

MASS 170 [M+]+ 170 [M+H]+ (Abd El Azim)

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.

Figure 6.32 Structure of Gallic acid

Introduction to gallic acid

Gallic acid (also known as 3,4,5-trihydroxybenzoic acid) is a trihydroxybenzoic acid, a type
of phenolic acid, found in gallnuts, sumac, witch hazel, tea leaves, oak bark, blueberries,
apples, flaxseeds, walnuts and other plants.
Gallic acid has been isolated from a number of plants, some of them are Allan blackia
floribunda, Garcinia densivenia, Bridelia micrantha, Caesalpinia sappan, Dillenia indica,
Diospyros cinnabarina, Paratecoma peroba, Psidium guajava, Syzygium cordatum, Rhus
typhina, Tamarix nilotica, Vitis vinifera, Hamamelis virginiana, Toona sinensis Oenothera
bienni and Rubus suavissimus (Nayeem et al.).
Physicochemical properties of gallic acid

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 Separation, Isolation and Characterization

Molecular weight: 170.12gm/mole

Molecular formula: C7H6O5
Colour: Yellowish-white
Solubility: Soluble in water, methanol, ether, glycerol, and acetone
(https://www.rsc.org/Merck-Index/monograph/m5645/gallic%20acid)
Pharmacological properties
Gallic acid posses anti–cancer, anti-inflammatory, antidiabetic, antihypertensive, antifungal,
diuretic, antiviral, anti- oxidant, anti-parkinson, antibacterial (Kasture et al.)
Storage condition
It is recommended to store at low temperature (5°C) (https://www.rsc.org/Merck
Index/monograph/m5645/gallic%20acid).
CONCLUSION
Chemical investigation of methanol extract of O. basilicum seed reveals presence of phenolic
and flavonoids. In present chapter, column chromatography method has been developed for
separation of phytoconstituent from methanolic seed extract of O. basilicum. After isolation
and identification of two phenolic compounds gallic acid, catechin, and one flavonoids
apigenin separated by column chromatography using toluene–acetone–formic acid as a
solvent. The structures of these compounds were confirmed by MP., UV, IR, MS, 1 H- NMR
13
and C-NMR spectral data. Above three compounds are reported first time from seed of O.
basilicum.
REFERENCES
Abd El Azim, Mohamed HM. “Phenolic Compounds and Cytotoxic Activities of Methanol
Extract of Basil (Ocimum Basilicum L.).” Journal of Microbial & Biochemical
Technology 7.4 (2015): 182–185.
Ashford, R.D. Ashford’s Dictionary of Industrial Chemicals. London, England: Wavelength
Publications Ltd, 1994.
“Catechin | The Merck Index Online.” 8 Apr. 2017.
Franz Bucar a, Abraham Wubea and Martin Schmidb. “Natural Product Isolation – How to
Get from Biological Material to Pure Compounds.” Natural product reports 13 (2013):
525–545.
Ghayur MN, Khan H, Gilani AH. “Antispasmodic, Bronchodilator and Vasodilator Activities
of (+)-Catechin, a Naturally Occurring Flavonoid.” Archives of Pharmacal Research
30.7 (2007): 970–975.
(https://www.rsc.org/Merck-Index/monograph/m5645/gallic%20acid)

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Kasture, Veena S. et al. “Antioxidant and Antiparkinson Activity of Gallic Acid Derivatives.”
Pharmacologyonline 1 (2009): 385–395.
Liu, Rui et al. “Synthesis and Biological Evaluation of Apigenin Derivatives as Antibacterial
and Antiproliferative Agents.” molecules 18 (2013): 11496–11511.
Nayaka, Hanumantappa B et al. “Antibacterial Attributes of Apigenin , Isolated from
Portulaca Oleracea L . Antibacterial Attributes of Apigenin , Isolated from.”
International Journal of Bacteriology October 2015 (2014): 1–8.
Nayeem, Naira et al. “Gallic Acid: A Promising Lead Molecule for Drug Development.”
Journal of Applied Pharmacy 8.2 (2016): 8–11.
Parikh, Nisha H, and Charmy S Kothari. “Development and Validation of a High-
Performance Thin-Layer Chromatographic – Densitometric Method for the
Quantification of Apigenin in Ocimum Basilicum.” Journal of Planar Chromatography
29.3 (2016): 216–220.
Ramasubramania, R. R., V. Sathyanathan, and C. Sekhar, V. and Roosewelt. “Standardization
and Antibacterial Screening of Ocimum Basilicum (Lamiaceae ) Leaf , Seed and Stem
Extracts.” International Journal of Pharmacy and Industrial Research 2.4 (2012): 440–
445.
S. Budavari. The Merck Index. 13th ed. New York: An Encyclopedia of Chemicals , Drugs
and Biologicals, 2006.
Sanjeev Shukla, Sanjay Gupta. “Apigenin: A Promising Molecule for Cancer Prevention.”
Pharmaceutical Research 27.6 (2010): 962–978.
Sasidharan S, Y Chen, D Saravanan. “Extraction, Isolation and Characterization of Bioactive
Compounds from Plants’ Extracts.” African Journal of Traditional, Complementary and
Alternative Medicines 8.1 (2011): 1–10.
Shashank, K, and KP Abhay. “Chemistry and Biological Activities of Flavonoids: An
Overview.” The Scientif World J 4.2 (2013): 32–48.
Yilmaz, Yusuf. “Novel Uses of Catechins in Foods.” Trends in Food Science & Technology
17 (2006): 64–71.

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