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CHAPTER 3
‘The important thing is not to stop questioning.’
~Albert Einstein
Purification and characterization of medicinally important
compounds isolated from different extracts of Indian spices
3.1 Rational
The exploitation of plants for secondary metabolites which contains
various important medicinal compounds has a long and valuable history since
at one time all drugs were obtained from natural sources. Even today plant
derived compounds play an important role in drug discovery for the curing of
many diseases (Cragg et al., 1997). It was estimated that the approximately
60% of the anti-tumor and anti-infective agents that are commercially
available are of natural origin.
Today many new technologies have been used which has powerful
result such, as high throughput screening and combinatorial synthetic
chemistry increase the possibility of drug discovery drastically. But natural
products still offer unmatched structural variety. A statistical investigation into
the structural complementarities of natural products and synthetic compounds
revealed to the investigated synthetic compounds. The potential for new
natural products still represent an important source for the lead finding process
of novel compounds which can be used as a possible source of therapeutic
(Bertel et al., 1999; Henkel et al., 1999; Verdine et al., 1996).
The natural products with the broadest range of therapeutic
applications are generally obtained from plant kingdom although many
naturally occurring drugs may be obtained from animals, microorganisms and
marine living resources (kinghorn, 1994). The potential of higher plants as
sources of new drugs is still largely unexplored. Among the estimated
250,000-50,000 plant species, a very small percentage has been investigated
phytochemically and the fractions submitted to biological or pharmacological
screenings is even smaller (Hamburger and Hostettmann, 1991: Harborne,
1998). It is apparent that drug discovery from plants is hindered by a number
of problems common to all such programs dealing with organisms, including
the often restricted supply of resource material, the biological variation of
different batches of collected plant material, and the need to rapidly
dereplicate active compounds of known structures (Kinghorn, 1994).
Now a day’s phytochemical research has been greatly facilitated by
the use of modern physio-chemical techniques of isolation and structure
elucidation. It has become easier to detect different classes of compounds,
such as alkaloids, and terpenoids (including cyclo-pentanoids, monoterpenes,
sesquiterpenes and other higher classes) by the modern techniques like column
chromatography , thin layer chromatography, high pressure liquid
chromatography, IR, mass, gas chromatography-mass spectrometry (GC-MS),
X-ray crystallography, and nuclear magnetic resonance (NMR) spectroscopy
(Derome, 1987; Atta-ur-Rahman, 1989).
3.2. Review of Literature
3.2.1 Nigella sativa L . (kalonji)
Specific chemical analyses of the volatile oil of Nigella sativa
L.started during the years 1960-1963 by Mahfouz and El-Dakhakhny (1960)
and Canonica et al., (1963). Enomoto et al., (2001) isolated the compound
from the methanol soluble portion of N. sativa L. as brown oil and was
analyzed for C11H16O3 in the high resolution-electron impact ionization (HR-
EI) mass spectrum. With 13C-NMR, distortion less enhancement by
polarization transfer (DEPT),1H–1H correlation spectroscopy (COSY), 1H–
13C COSY, Nuclear Overhauser Enhancement and Exchange Spectroscopy
(NOESY), and Heteronuclear Multiple Bond Correlation (HMBC) spectra,
was determined to be 2-(2-methoxypropyl)-5-methyl-1,4-benzenediol.
Taskin et al., (2005) reported three known triterpene glycosides which were
isolated from the MeOH extract of dried and powdered seeds of Nigella sativa
L. The structures of the compounds were established as 3-O-[_-D-
xylopyranosyl-(1/3)-L-rhamnopyranosyl-(1/2)-L-arabinopyranosyl]-28-O-[_-
Lrhamnopyranosyl-(1/4)-D-glucopyranosyl-(1/6)-D glucopyranosyl]-
hederagenin, 3-O-[_-L-rhamnopyranosyl-(1/2)-L-arabinopyranosyl]-28-O-[_-
L- rhamnopyranosyl-(1/4)-D-glucopyranosyl-(1/6)-D-glucopyranosyl]-
hederagenin and 3-O-[_-D-xylopyranosyl-(1/3)-L-rhamnopyranosyl-(1/2)-L-
arabinopyranosyl]–hederagenin.
Philipov et al., (2004) obtained the tertiary and quaternary alkaloid
from the ethanol extract of a whole plant of N. arvensis. Eight known
aporphine alkaloids were isolated from the alkaloid fractions by
chromatographic procedures. The structures of the alkaloids were elucidated
by direct comparison of their Rf values, IR, MS and 1H NMR spectral data
with those of the authentic samples. The main alkaloids are glaucine
(Kuzmanov et al., 1992) and oxoglaucine (Philipov et al., 1998). The other
alkaloids are predicentrine (Philipov et al., 1983), bracteoline (Mollov and
Philipov, 1979), isoboldine (Guinaudeau et al., 1975), N-methylglaucine
(Guinaudeau et al., 1983), N-methyllaurotetanine (Kande et al., 1994), and
asimilobine (Philipov et al., 2000).
Rahman et al., (1995) reported the nigellidinea a new indazole alkaloid
from the seeds of Nigella. The rare indazole-type alkaloid nigellidine is
accompanied by its 4-O-sulfite in the seeds of Nigella sativa L. was reported
by Ali et al., (2008) compound may represent the true natural product leading
to nigellidine via hydrolysis of the sulfate functionality during the isolation
process. The structure of nigellidine-4-O-sulfite is confirmed by NMR, MS,
and X-ray crystallographic data. These studies were complemented by most
recent ones which revealed various pharmacologically active constituents that
included Thymoquinone (2-isopropyl-5-methyl-benzoquinone) that may attain
up to 27.8% of the volatile oil (Houghton, 2006; Canonica, 1963; Burits,
2000), Carvacrol (2-methyl-5-(1-methyl ethyl) phenol which is also known as
2-hydroxy-p-cymene or isothymol) (5.8-11.6%) , p-cymene (isopropyl
toluene) in the range of 15.5-31.7% (Burits, 2000; Aboutabl, 1986), α-pinene
(2,6,6-trimethylbicyclo [3.1-1]-hepta-2-ene (9.3%) (Aboutabl, 1986), 4-
terpineol (or α -terpineol or α, α,4-trimethyl-3-cyclo-hexene-1-methanol or p-
menth-1en-8-ol) 2-6.6% longifolene (or Junipene or Kuromatsuene or
decahyro-4,8,8-trimethyl-9-methylene-1,4-methanoazulene) 1-8% (Burits
2000), t-anethole (p-Propenyl anisole or 1-methoxy-4-(1-propenyl) benzene
0.25-2.3% (Burits 2000) and the reduction product of thymoquinone
thymohydroquinone together with some esters about 16% (Aboutabl, 1986).
Kapoor et al., (1990) isolated esters of fatty acids, e.g., oleic acid,
linoliec acid, and dehydrostearric acid, higher terpenoids, aliphatic alcohols,
and α-β unsaturated hydroxyl ketones from Nigella sativa L. seeds. The active
constituents of the seeds include the volatile oil consisting of carone, and
unsaturated ketone, terpenes as d-limonene, carnone and cymene. Free sterols,
steryl esters, steryl glucosides and acylated steryl glucosides were isolated
from the seed oil (Menounos, 1986).
A novel alkaloid, nigellicine, an isoquinoline alkaloid, nigellimine, and
an indazole alkaloid, nigellidine, were also isolated from the seeds of Nigella
sativa L. (Rehman, 1985). The crystalline active principle, nigellone, is the
only constituent of the carboxyl fraction of the oil. Pharmacologically active
constituents of the volatile oil are thymoquinone, dithymoquinone,
thymohydroquinone, and thymol (Ghosheh, 1999).
3.2.2. Piper nigrum L. (Black pepper)
Aldaly (2010) reported a pungent alkaloid, named as Piperine and was
characterized as [1-[5-[1, 3-benzodioxol-5-yl]-1-oxo-2, 4, pentadienyl
piperidine. Navickiene et al., (2000) reported the isolation of number of
amides bearing isobutyl, pyrrolidine, dihydropyridone and piperidine moieties
from Piper hispidum and Piper tuberculatum .The isolation and
characterization of several representatives including two hitherto unreported
amides were performed by chromatographic techniques and by analysis of
spectroscopic data.
From chloroform extract of black pepper, Park et al., (2002) isolated
four N-isobutylalamine alkaloids i.e., Pellitorine, guineesine, pipericide and
retrofractamide A and one piperidine alkaloid i.e., piperine through column
chromatography and identified through spectroscopy.
Alecio et al., (1998) reported the isolation of pyrrolidine amide, N-[7-
(3´,4´- methylenedioxyphenyl)-2(Z), 4(Z)-hepadienoyl] pyrrolidine, in
addition to two amides N-[5-(3´,4´-methylenedioxyphenyl)-2(E)-
pentadienoyl] pyrrolidine and N-[2-(3´,4´-methylenedioxy-6-methoxyphenyl)-
2(Z)-propenoyl] pyrrolidine from chloroform extract from leaves of Piper
hispidium. The structure of was elucidated by interpretation of spectral data,
including ES-MS.
Three bisalkaloids, dipiperamides were also reported from the white
pepper (Piper nigrum L.) along with the known piperine and piperylin
(Tsukamoto, 2002).
3.2.3. Pimpinella anisum L . (Aniseed)
Eight glycosides of 2-C-methyl-D-erythritol (1) were isolated from the
fruit of anise, and their structures were clarified as 1-O-β- D -glucopyranoside,
3-O-β-D-glucopyranoside, 4-O-β- D -glucopyranoside, 1-O-β- D -
fructofuranoside, 3-O-β-D-fructofuranoside,4-O-β-D-fructofuranoside,1-O-β-
D-(6-O D 4hydroxybenzoyl)-glucopyranoside and 1-O-β-D-(6-O-4-
methoxybenzoyl)-glucopyranoside of 2-C-methyl- D -erythritol respectively
(Kitajima et al., 2003).
Baser et al.,(2007) reported the isolation of [4-(prop-2-enyl)phenyl
angelate and 4-(3-methyloxiranyl)phenyl 2-methylbutyrate], bisabolene type
sesquiterpenoid [1-methyl-4-(6-methylhepta-1,5-dien-2-yl)-7-
oxabicycloheptane (aureane)] and trinorsesquiterpene [4-(6-
methylbicyclohept-2-en-7yl)butan-2-one (traginone)] from the essential oils
of Pimpinella species occurring in Turkey. Pimpinella essential oils were
subjected to column chromatography (silica gel) using n-hexane and diethyl
ether .Structure elucidation of the isolated compounds was achieved by a
combination of 1D and 2D NMR techniques. From the pimpinella oil γ-
Himachalene and the diterpene neophytadiene were isolated by TLC and
column chromatography at low temperatures. Their structures were
determined by MS and NMR including1H-1H correlated COSY and NOESY
by Burkhardt et al., (1986).
3.2.4. Trachyspermum ammi L. (Ajwain)
Mathew et al., (2008) reported that the, the yields of the methanolic
crude residue after removal of the solvent from the T. ammi L. extract and the
active fraction after silica gel column chromatography (SGCC) were 14.15%
w/w and 5.76% w/w respectively. The residue of the crude extract was
brownish coloured oily liquid. On repeated chromatographic purification the
active fraction yielded a white crystalline solid that by combined FTIR, NMR
and mass spectral analysis was identified as 2-isopropyl-5-methyl phenol. GC
and GC-MS analysis of ajwain essential oil showed the presence of 26
identified components which account for 96.3% of the total amount. Thymol
(39.1%) was found as a major component along with p-cymene (30.8%), β-
terpinene (23.2%), β-pinene (1.7%), terpinene-4-ol (0.8%) whereas acetone
extract of ajwain showed the presence of 18 identified components which
account for 68.8% of the total amount. The major component was thymol
(39.1%) followed by oleic acid (10.4%), linoleic acid (9.6%), α-terpinene
(2.6%), p-cymene (1.6%), palmitic acid (1.6%), and xylene (0.1%). Garg and
Kumar, (1998) reported the alkaloid from the methanolic extract of ajwain
which contains 6-O- β-D glucopyranosyloxythy.
3.3 Materials and Methods
Different extracts of considerable interests of N. sativa L., P. nigrum L., P
.anisum L. and T. ammi L. were subjected to column chromatography.
3.3.1 Column chromatography
3.3.1.1 Preparation of slurry: - Slurry of the extracts of all the four spices
i.e., N. sativa L. (methanol extract), P. nigrum L. (ethyl acetate
extract), P. anisum L. (chloroform extract) and T. ammi L. (methanol
extract) were made. According to the weight of extracts, of N. sativa
L. (methanol extract), P. nigrum L. (ethyl acetate extract), P .anisum L.
(chloroform extract) and T. ammi L. (methanol extract) that is 5.0 gm,
3.5 gm, 4.0 gm and 5.5gm respectively, the ten times silica was added
and mixed properly by adding small amount of petroleum ether, the
slurry was neither to dry nor to wet.
3.3.1.2 Column Packaging:- Column was filled with silica (pore size 60-120
mesh) the 30 times the weight of extract and setelled with petroleum
ether, while filling the silica in the column it was thaw properly to
avoid the air bubble. Slurry of the extract was made in petroleum ether
and was poured over the well setelled column and was digested by
leaving the column for ½ an hour.
3.3.1.3 Elution of Column to obtained fraction:- These columns were eluted
with different solvents like petroleum ether, benzene, chloroform, ethyl
acetate, methanol and their mixtures of increasing polarities. Several
fractions were obtained which was monitored with TLC. Similar
fractions of identical Rf value were pooled together. These pooled
fractions on vacuum concentration affored considerable solid mass.
3.3.2 Thin layer chromatography (Wagner, R., and Bladt, S., 1996)
3.3.2.1 Preparation of TLC Plate:- The TLC silica was used to make the
slurry. The silica was mixed with distilled water to make a gelatinous
mass, mixed properly to avoid the clump formation. Using a soft
pencil, light line was drawn along the plate 1 cm from one edge to spot
samples along this line. Then marked darker 0.5 cm lines on this line
to indicate sample position. A small amount of slurry was put on a well
dry & clean glass slide and it was equally distributed throughout the
slide with the help of glass slide.
3.3.2.2 Activation of TLC Plate :- The freshly prepared TLC Plate after few
minutes were kept in the hot air oven at 100 °C for one to two hour
for the activation.
3.3.2.3 Equilibration of TLC Plate: Equilibrated the TLC tank with
developed solvent of the polarity according to the fraction solubility,
(which is eluted through column chromatography) for ~ one hour.
Typically 10-15mL of solvent is used.
3.3.2.4 Loading of the Sample:- The sample was dissolved in organic
solvent according to its solubility a small drop of sample was loaded
on the line 1cm from the edge with the help of small glass capillary
tube and allow to dry for apporx. 15 min.
3.3.2.5 Running of the sample:- The charged TLC plate was put in the TLC
tank for the separation (if more then one compound is present in the
fraction). Open the tank and gently put the plate in, samples down.
Splashing into the solvent was avoided and prop the bottom edge
against the glass spine and the top edge against the filter paper liner
was propped and removed the plate when it reaches solvent front line.
3.3.2.6 Analysis of the Data:- After removing the TLC plate from the TLC
tank it was kept at room temperature for 1 hour , for proper drying. The
iodine solution was sprayed for the visualization of spot and if the spot
was not clear, the TLC plate was visualized under the UV chamber at
245nm.
3.3.2.7 Calculation for Retention factor
Rf value = Distance travelled by the solute (cm) Distance travelled by the solvent (cm)
3.3.3 Spectroscopic Analysis Out of these fractions, the fraction of same Rf value were mixed together
and some fractions were crystallized. The pure crystallized compounds were
characterized by melting point, solubility and spectral studies like I.R, NMR
(1H, 13C) and mass spectral studies. All these studies were carried out at
Sophisticated Analtytical Instrument Facility (SAIF), CDRI, Lucknow.
3.4 Results and Discussions:-
The results of this chapter were divided into two parts according to the
purification and characterization of the isolated secondary metabolites
from the spices used in the present study.
1. Column chromatography and TLC (Purification)
2. Spectroscopic Analysis of the purified compounds.
From the methanolic extract of Nigella sativa L. seed a new oleane
triterpenoid has been isolated which is characterized as 3β- hydroxy-olean-12
(13)-ene-28-oic acid. From the ethyl acetate extract of Piper nigrum L. an
amide has been purified which is named as 3-(3, 4-dihydroxyphenyl)-N-[2-
(4-isopropyl phenyl)-ethyl] acrylamide where as from the chloroform extract
of Pimpinella anisum L. and methanol extract of Trachyspermum ammi L. the
purified compounds are characterized as 4-(2-propenyl) phenyl isobutyrate
and Olean-12-ene-3β-ol respectively. The detailed of the fraction eluted during
chromatography and spectral values are as follows.-
1. Column Chromatography and Thin layer Chromatography
Table 3.1: Data of column Chromatography and TLC of methanolic extract of N. sativa L. (the criteria for selecting the solvent system was polarity).
S.No. Solvent system used No. of Fractions Collected
Result of TLC(mixture/No. of
spot) 1 Petroleum Ether 1 – 8 mixture
2 Pet. : Benzene 10:1 9 – 11 mixture
3 10:02 12 – 14 mixture
4 10:03 15 – 18 mixture
5 10:04 19 – 22 mixture
6 10:05 23 & 24 mixture
7 10:06 25 & 26 two
8 10:07 27- 30 two
9 10:08 31 – 34 two
10 10:09 35 – 37 two
11 Benzene pure 38 – 40 mixture
12 Ben. : Chloroform 10 : 1 41 – 44 mixture
13 10:02 45 – 58 Single
14 10:03 59– 62 mixture
15 10:04 63 – 68 mixture
16 10:05 69 – 72 mixture
17 10:06 73 – 75 mixture
18 10:07 76 – 79 two
19 10:08 80 – 84 two
20 10:09 85 – 90 two
21 chloroform : Acetone 10 : 1 91 – 95 two
22 10:02 96 – 99 two
23 10:03 100 – 102 mixture
24 10:04 103 – 106 mixture
25 10:05 107 – 110 mixture
26 10:06 111 – 113 mixture
27 10:07 114 – 117 mixture
28 10:08 118 -120 two
29 10:09 121 – 127 two
30 Acetone : Methanol 10 : 1 128 – 135 two
31 10:02 136 -138 two
32 10:03 139 – 144 two
33 10:04 135 – 150 two
34 10:05 151 – 153 two
35 10:06 154 – 157 mixture
36 10:07 158 – 161 mixture
37 10:08 162 – 165 mixture
38 10:09 166 – 168 9*mixture
39 Methanol Pure 169 – 189 mixture
3.4.1 Nigella sativa L.
The methanolic extract of Nigella sativa L. was considered for further
investigation. Slurry was made to this 5.0 gm mass with 50 gm of silica gel
with pore size 60-120 mesh (Fig. 3.1). This slurry was loaded to a column of
silica gel (150 gm) well settled in petroleum ether. After the digestion of mass,
the column was eluted with petroleum ether, benzene, chloroform, ethyl
acetate, methanol and their mixtures of different ratios of increasing polarity
depending on which type of compound is analyzed (Emilio, 2008 ). The total
170 different fractions (Table 3.1) of eluent were collected (Fig 3.5) and
monitored with TLC. Similar fractions of identical Rf value were pooled
together. The fraction obtained by eluting the column benzene-chloroform
(5:1) solvent system with Rf value of 0.83, afforded the separation of
amorphous powder, which was recrystallized by methanol to give white
coloured shiny micro needles (24 mg) with melting point 298-300°C.
Enomoto et al., (2001) isolated the compound from the methanol soluble
portion of N. sativa L. Taskin et al., (2005) reported three known triterpene
glycosides which were isolated from the MeOH extract of dried and powdered
seeds of Nigella sativa L. Kapoor et al., (1990) isolated esters of fatty acids,
e.g., oleic acid, linoliec acid, and dehydrostearric acid, higher terpenoids,
aliphatic alcohols, and α-β unsaturated hydroxyl ketones from Nigella sativa
L. seeds.
Table 3.2: Data of Column Chromatography and TLC of ethyl acetate extract of P. nigrum L.
S. no. Solvent system used No. of Fractions Collected
Result of TLC(mixture/No.
of spot) 1 Petroleum Ether 1 – 10 mixture
2 Pet. : Benzene 10:1 11 – 15 mixture
3 10:02 16 – 18 mixture
4 10:03 19 – 21 mixture
5 10:04 22 – 25 mixture
6 10:05 26 & 27 mixture
7 10:06 28-30 two
8 10:07 31- 33 two
9 10:08 35 – 38 two
10 10:09 39 – 40 two
11 Benzene pure 41-44 mixture
12 Ben. : Methanol 10 : 1 45 – 48 mixture
13 10:02 49 – 52 mixture
14 10:03 53 – 56 mixture
15 10:04 57 – 65 mixture
16 10:05 66 – 69 mixture
17 10:06 70 – 75 mixture
18 10:07 76 – 79 two
19 10:08 80 – 84 two
20 10:09 85 – 89 mixture
21 Chloroform: Methanol 10: 1 90 – 103 Single
22 10:02 104 – 108 two
23 10:03 109 – 112 mixture
24 10:04 113 – 116 mixture
25 10:05 117 – 120 mixture
26 10:06 121 – 123 mixture
27 10:07 124 – 127 mixture
28 10:08 128 -131 two
29 10:09 132 – 137 two
table continued………
30 Acetone : Methanol 10 : 1 138 –140 two 31 10:2 141 -143 two 32 10:3 144 –148 two 33 10:4 149&150 two 34 10:5 151–154 two 35 10:6 155 –160 mixture 36 10:7 161–167 mixture 37 10:8 168– 175 mixture 38 10: 9 176 – 179 mixture 39 Methanol Pure 188 – 207 mixture
3.4.2. Piper nigrum L.
The ethyl acetate extract of Piper nigrum L. was considered for further
examination to obtained pure compound .Slurry was made to this mass (3.5
gm) with small amount of silica gel (35 gm). This slurry was loaded to a
column of silica gel (110 gm) well settled in petroleum ether (Fig. 3.2). After
the digestion of mass the column was eluted with petroleum ether, benzene,
chloroform, ethyl acetate, methanol and their mixtures of different ratios of
increasing polarity depending on which type of compound is analyzed
(Emilio, 2008 ).. 190 different fractions (Table 3.2) of eluent were collected
(Fig. 3.6) and monitored with TLC. Similar fractions of identical Rf value were
pooled together. The fractions (90-93) obtained by eluting the column with
chloroform-methanol (10:1) solvent system with Rf value 0.52 afforded the
separation of amorphous powder, which was recrystallized from acetone-
methanol (1:5) to give fine needle shaped yellow coloured crystal. Navickiene
et al., (2000) reported the isolation of number of amides bearing isobutyl,
pyrrolidine, dihydropyridone and piperidine moieties from Piper
hispidum and Piper tuberculatum. From chloroform extract of black pepper,
Park et al., (2002) isolated four N-isobutylalamine alkaloids i.e., Pellitorine,
guineesine, pipericide and retrofractamide A and one piperidine alkaloid i.e.,
piperine through column chromatography.
Table 3.3: Data of Column Chromatography and TLC of Chloroform extract of P. anisum L.
S.no Solvent system used No. of Fraction Collected
Result of TLC(mixture/No. of spot)
1 Petroleum Ether 1 – 8 mixture
2 Pet. : Benzene 9:1 9 – 11 mixture
3 08:02 12 – 14 mixture
4 07:03 15 – 18 mixture
5 06:04 19 – 22 mixture
6 05:05 23 & 24 mixture
7 04:06 25 & 26 two
8 03:07 27- 30 two
9 02:08 31 – 34 two
10 01:09 35 – 37 two
11 Benzene pure 38 – 40 mixture
12 Ben. : Chloroform 9 : 1 41 – 44 mixture
13 08:02 45 – 47 mixture
14 07:03 48 – 52 mixture
15 06:04 53 – 58 mixture
16 05:05 59 – 62 mixture
17 04:06 63 – 65 mixture
18 03:07 66 – 69 two
19 02:08 70 – 74 two
20 01:09 75 – 80 two
21 Chloroform: Acetone 9 : 1 81 – 85 two
22 08:02 86 – 89 two
23 07:03 90 – 92 mixture
24 06:04 93 – 96 mixture
25 5:5 97 – 100 mixture
26 04:06 101 – 103 mixture
27 03:07 104 – 107 mixture
28 02:08 108 -110 two
29 01:09 111 – 117 mixture
table continued……
30 Acetone: Methanol 9 : 1 118 – 125 two
31 08:02 126 -128 two
32 07:03 129 – 134 two
33 06:04 135 – 140 two
34 05:05 141 – 143 two
35 04:06 144 – 160 single
36 03:07 161 – 167 mixture
37 02:08 168 – 175 mixture
38 1: 9 176 – 183 mixture
39 Methanol Pure 184– 211 mixture
3.4.3. Pimpinella anisum L .
The chloroform extract (4.0 gm) of Pimpinella anisum L. was
considered for further investigation to get pure compound. Slurry was made to
this mass with 40 gm of silica gel. This slurry was loaded to a column with
120 gm silica gel (Fig. 3.3) well settled in petroleum ether. After the digestion
of mass the column was eluted with organic solvent like, petroleum ether,
benzene, chloroform, ethyl acetate, methanol and their mixtures of different
ratios of increasing polarity depending on which type of compound is
analyzed (Emilio, 2008 ). Total 190 different fractions (Table 3.3) of eluent
were collected (Fig3.7) and monitored with TLC. Similar fractions of identical
Rf value were pooled together. The fraction obtained by eluting the column
with acetone-methanol (4:6) solvent system afforded the separation of
amorphous powder with Rf value of 0.58, which was recrystallized from
dichloromethane: ethylacetate (1:2) to give needles shaped crystals. Zhi (1999)
reported three alkaloids through column chromatography using solvent system
of increasing polarity from the roots of P. thellungiana.
Table 3.4: Data of Column Chromatography and TLC of Methanolic extract of T. ammi L.
S. no Solvent System used No. of Fractions Collected
Result of TLC(mixture/No.
of spot) 1 Petroleum Ether 1 – 7 mixture
2 Pet. : Benzene 10:1 8 – 11 mixture
3 10:02 12 – 18 mixture
4 10:03 19 – 24 mixture
5 10:04 25 – 29 mixture
6 10:05 30 -33 mixture
7 10:06 34 – 39 two
8 10:07 40- 42 two
9 10:08 43 – 47 two
10 10:09 48 – 51 two
11 Benzene pure 52 – 55 mixture
12 Ben. : Chloroform 10 : 1 56 – 58 mixture
13 10:02 59 – 73 single
14 10:03 74 & 75 mixture
15 10:04 76 – 80 mixture
16 10:05 81 – 89 mixture
17 10:06 90 – 95 mixture
18 10:07 96 – 99 two
19 10:08 100 – 104 two
20 10:09 105 – 109 Mixture
21 Chloroform: Acetone 10 :1 110 – 113 two
22 10:02 114 – 118 two
23 10:03 119 – 122 Mixture
24 10:04 123 – 126 mixture
25 10:5 127 – 130 mixture
26 10:06 131 – 133 mixture
27 10:07 134 – 137 mixture
28 10:08 138 -141 two
29 10:09 142 – 147 two
table continued………
30 Acetone : Methanol 10 : 1 148 – 150 two
31 10:02 151 -153 two
32 10:03 154 – 158 two
33 10:04 159 & 160 two
34 10:05 161 – 164 two
35 10:06 165 – 167 mixture
36 10:07 168 – 161 mixture
37 10:08 162 – 165 mixture
38 10:9 166 – 168 mixture
39 Methanol Pure 169 – 190 mixture
3.4.4. Trachyspermum ammi L.
The methanol extract of Trachyspermum ammi L. (5.5 gm) was
considered for further studies. Slurry was made to this mass with 55gm of
silica gel. This slurry was loaded to a column with 165 gm of silica gel well
settled in petroleum ether (Fig. 3.4). After the digestion of mass the column
was eluted with petroleum ether, benzene, chloroform, ethyl acetate, methanol
and their mixtures of different ratios of increasing polarity depending on
which type of compound is analyzed (Emilio, 2008 ). Total 180 different
fractions (Table 3.4) of eluent were collected (Fig. 3.8) and monitored with
TLC. Similar fractions of identical Rf value were pooled together. The fraction
obtained by eluting the column with benzene- chloroform (5:1) solvent system
afforded the separation of amorphous powder with Rf value 0.69, which was
recrystallized from acetone to give micro needles. Mathew et al., (2008)
reported that the, the yields of the methanolic crude residue after removal of
the solvent from the T. ammi L. extract and the active fraction after silica gel
column chromatography (SGCC). Garg and Kumar, (1998) reported the
alkaloid from the methanolic extract of ajwain which contains 6-O- β-D
glucopyranosyloxythy.
(Fig. 3.1) (Fig. 3.2)
(Fig .3.3) (Fig. 3.4)
Fig. 3.1: Column Chromatography of N. sativa L.
Fig. 3.2: Column Chromatography of P. nigrum L.
Fig. 3.3: Column Chromatography of P. anisum L.
Fig. 3.4: Column Chromatography of T. ammi L.
(Fig. 3.5) (Fig. 3.6)
(Fig. 3.7) (Fig. 3.8)
Fig. 3.5: Fractions eluted through Column Chromatography of N. sativa L.
Fig. 3.6: Fractions eluted through Column Chromatography of P. nigrum L.
Fig. 3.7: Fractions eluted through Column Chromatography of P. anisum L.
Fig. 3.8: Fractions eluted through Column Chromatography of T. ammi L.
2. Spectroscopic analysis
3.4.5. Nigella sativa L.
3ββββ- hydroxy-olean-12 (13)-ene-28-oic acid.
The colourless amorphous powder obtained from the methanol extract
by eluting the column with benzene- chloroform (5:1) showed positive
Liebermann Burchard test for characteristic unsaturated triterpenoid. The
absorption bands observed at 3460, 1690 and 1630 cm-1 in its I.R spectrum
(Table 3.5.1) indicated the presence of hydroxyl, carboxylic and olefinic
linkage in the molecule.
The 1H NMR- spectrum (Table 3.5.2) of the compound observed a
one-proton multipet at δ 5.24 was assigned to olefinic proton H-12. Another
one proton double doublet at δ 3.45 was clearly assignable to α- methine H-3
proton. In addition, the spectrum also observed three singlet’s for seven
tertiary methyl groups between1.09 to 0.99 (Fig. 3.9). The presence of these
functionalities was further supported by 13 C NMR spectrums (Table 3.5.3).
The downfield signal appeared at δ 179.5 was assignable to C-28 carboxylic
group. Another downfield signals observed at δ 124.1 and δ 143.6 were due to
olefinic bond between C12 -C13. The signal for C-3 alpha proton was observed
at δ 88.5.
These spectral data were well in support of β- amyrin type structure of
the compound. By analogy with acid of β- amyrin group, the position of
hydroxyl group was assumed to be at C-3 in ring A, carboxylic group at C-17
and carbon- carbon double bond between C12 – C13. The structure of the
compound was further confirmed by the mass fragmentation pattern (Table
3.5.1) seen in its mass spectra. The molecular ion peak [M+] at m/z 456
corresponded to the molecular formula C30H48O3. The other characteristic
peaks observed at m /z 248 and 208 were due to the rings A/B and D/E arose
due to C9,11 – C8,14 cleavage. The other important peaks at m/z 203 and 190
were due to loss of carboxylic acid and water molecule from their respective
fragments. On the basis of the above spectral studies the structure of isolated
compound was assigned as 3ββββ- hydroxy-olean-12 (13)-ene-28-oic acid.
Mass fragmentation Pattern of 3ββββ- hydroxy-olean-12 (13)-ene-28-oic acid.
C9,11- C8,14 Cleavage
m/z 208 m/z 248
m/z 190 Removal of H2O m/z 203 Removal of COOH
3.4.6. Piper nigrum L.
3-(3, 4-dihydroxyphenyl)-N-[2-(4-isopropyl phenyl)-ethyl] acrylamide.
The compound was obtained as needle shaped yellow coloured crystals
(20mg) from the ethyl acetate extract by eluting the column with chloroform-
methanol (10:1) and recrystallized from acetone-methanol (1:5). The
compound had a molecular composition C20H23NO3 assigned on the basis of
chemical analysis and molecular ion peak observed at m/z 325 in its mass
spectrum.
The IR spectrum of the compound showed the absorption bands indicating
highly conjugated system bearing amide (3260 cm-1), α, β-unsaturated
carbonyl (1650 cm-1), hydroxyl group (3540cm-1) and general methyl group
(1380,1360cm-1). (Table 3.6.1)
The 1H NMR spectrum of the compound (Table 3.6.2) displayed ABX-
Type signals of aromatic ring A proton at δ 6.68 (H-2), 6.56 (H-5) and 6.66
(H-6) along with olefinic proton signals at δ 6.63 (H-2) and δ 7.53 (H-3) were
indicative of the presence of a disubstituted cinnamate group in the compound.
The aromatic ring B proton signals appeared as two proton doublets at δ 7.02
and δ 6.97 attributable to H-2´/H-6´ and H-3´/H-5´ respectively. Two triplets
for two protons each at δ 3.28 and δ 2.79 were assigned to methylene proton
H-1´´ and H-2´´respectively. A one proton multiplet at δ 2.19 and two three
proton double at δ 1.00 and 0.96 were assigned to the methine and methylene
proton of isophenyl group at to C-4´of ring B (Fig. 3.10).
The 13 C NMR spectrum (Table 3.6.3) exhibited signals which further
helped in assigning these functionalities. A dominified signal at δ 166.4 was
assigned to carbonyl function at C-1 where as signals at δ 119.7 and 142.1
were ascribed to C-2 and C-3 carbon atoms or olefinic function. The phenolic
carbon atom of ring A signaled at144.2 and 143.7 bearing hydroxyl group and
of ring B C-4´ bearing isopropyl side chain resonated at δ 138.1.
The mass spectrum (Table 3.6.1) displayed a highly intensified peak at
m/z 163 which was generated due to loss of C11H16N fraction caused by C1-
NH cleavage. This peak along with at m/z 135 generated the C1-C2 cleavage
further supported the hydroxyl groups in ring A. Another peak appeared at m/z
282 was generated due to elimination of side chain C3H7. This fragmentation
pattern was further in support of the hydroxyl group attached to ring A and
side chain to ring B.
On the basis of above studies the compound were characterized as 3-
(3,4-dihydroxyphenyl)-N-[2-(4-isopropyl phenyl)-ethyl] acrylamide.
This compound on acetylation with acetic anhydride/ pyridine afforded
a diacetate product confirming the presence of two hydroxyl groups in the
compound. The 1H NMR spectrum of this compound showed a six proton
singlet at δ 2.07 for acetyl proton in addition to signals observed in non
acetoxy compound further in the evidence of a dihydroxy product.
Mass Fragmentation Pattern of 3-(3,4-dihydroxyphenyl)-N-[2-(4-
isopropyl phenyl)-ethyl] acrylamide
C1- NH Cleavage
m/z 135 mm/z 198 C1-C2 Cleavage
Removal of side
chain
m/z 282
3.4.7. Pimpinella anisum L.
4-(2-propenyl) phenyl isobutyrate
The compound was isolated as white amorphous powder from the
chloroform extract by eluting the column with acetone-methanol (4:6). The
molecular peak in its mass spectrum appeared at m/z 203 correspond to the
molecular composition C13H16O2. The spectral studies clearly indicated the
presence of carbonyl function, unsaturation and the phenyl group in the
molecule. The 1H NMR spectrum of the compound observed signals of 4H
proton in aromatic range between δ 7.01-6.90 clearly indicating the
disubstituted benzene ring in the molecule (Fig. 3.11). This was further
supported by 13C NMR spectrum which exhibited signals in the range 142-
129. Two signals for one proton each in downfield region at δ 5.21 & δ 5.29
clearly indicated the presence olefinic protons in the molecule. It was further
supported by 13C NMR showing resonance at 129 and 121 for these olefinic
carbon atoms. The presence of carbonyl function was supported by 13C NMR
which gave signal at 161.8. The compound also bears gem methyl group
which was supported by the appearance of 1H signal at δ 3.37 (CH) and at
1.08 -0.95 (6H, 2 x CH3). In13C NMR these resonances appeared at 45.2 and
23.8 respectively. On the basis of these observations the compound was
considered to be 4-(2-propenyl) phenyl isobutyrate.
3.4.8. Trachyspermum ammi L .
Olean-12-ene-3β-ol
The compound was obtained as colourless amorphous powder (20 mg)
from the methanol extract by eluting the column with benzene –chloroform
(5:1) and was recrystallized from acetone. The compound responded
Liebermann Burchard test, characterization of pentacyclic triterpene structure.
The pentacyclic triterpene structure was further supported by molecular
composition and molecular ion peak observed at m/z 426 corresponding to
C30H50O.
The IR spectrum (Table 3.7.1) of the compound showed the presence
of hydroxyl group at 3420cm-1 and olefinic linkage at 1630 cm-1. The 1H NMR
spectrum of the compound (Table 3.7.2) observed one proton multiplet at δ
5.24 clearly assignable to olefinic proton H-12. Another one proton multiplet
observed at δ 3.44 was assigned to carbonyl proton H-3. H-18 proton was
observed a multiplet at δ 3.22. The signals for all the eight methyl groups
appeared a singlet between δ 1.25-0.97 clearly indicated the attachment to
saturated carbon atoms (Fig. 3.12). The presence of hydroxyl group at C-3
and olefinic linkage between C-12 and C-13 was further supported by 13 C
NMR spectrums (Table 3.7.3) which exhibited domnfied signals at δ 126.0
and 141.3 assigned for C-12 and C-13 respectively.
The signal observed at δ 79.8 was assigned to carbonyl carbon C-3.
The characteristic peak observed at m/z 218 and 208 in its mass spectrum
(Table 3.7.1) were generated due to C9-C11 and C8-C14 cleavage. Another
diagnostic peak observed at m/z 411,408, 393, 203,193 and 188 further
supported the structure as Olean-12-ene-3β-ol
Table 3.5.1: Data of IR and Mass spectrum of Nigella sativa L.
1 Anal. Found C: 78.82 H: 10.49 2 Calcd.for C30H48O3 C: 78.95 H: 10.53 3 IR. (KBr) λmax 3460, 1690, 1630 cm-1
4 Mass spectrum m/z 456 [M+]; 248 [Ring C/D] ; 208 [Ring A/B] ; 203 [248- COOH] ; 190 [208- H2O]
Table 3.5.2: Data of 1H NMR chemical shift of 3β- hydroxy-olean-12 (13)-
ene-28-oic acid of Nigella sativa L.
S.no 1H NMR chemical shift Assingment 1 δ 0.95 (6H, s, 2 Me 24 &25) 2 δ 0.99 (6H, s, 2 Me 26 & 29) 3 δ 1.09 (9H, s, 3Me 23, 27 & 30) 4 δ 3.27 (1H, dd, H-18) 5 δ 3.45 (1H, dd, H-3) 6 δ 5.24 (1H, m, H-12)
Table 3.5.3: Data of 13C NMR chemical shift of 3β- hydroxy-olean-12 (13)-
ene-28-oic acid of Nigella sativa L. Carbon atom 13C NMR
chemical shift Carbon atom 13C NMR
chemical shift C-1 38.2 C-16 24.5 C-2 27.0 C-17 45.2 C-3 88.5 C-18 40.5 C-4 38.0 C-19 44.6 C-5 54.1 C-20 30.6 C-6 18.2 C-21 36.3 C-7 33.1 C-22 33.3 C-8 35.3 C-23 28.0 C-9 48.5 C-24 16.7 C-10 37.2 C-25 15.5 C-11 24.2 C-26 16.9 C-12 124.1 C-27 23.9 C-13 143.6 C-28 179.5 C-14 40.5 C-29 31.2 C-15 28.5 C-30 24.2
Table 3.6.1: Data of IR and Mass spectrum of Piper nigrum L.
1 Anal. Calcd C: 73.84; H: 7.07
2 for C20H23NO3 C: 74.16 : H:8.24
3 IR. (KBr) λmax 3540, 3260, 1650, 1380, 1360 cm-1
4 Mass Spectrum m/z 325[H+] C20H23NO3
282, 190, 163, 135.
Table 3.6.2: Data of 1H NMR chemical shift of 3-(3, 4-dihydroxyphenyl)-N-
[2-(4-isopropyl phenyl)-ethyl] acrylamide of Piper nigrum L.
S.no 1H NMR chemical shift No. of Proton multiplicity
Assingment
1 δ 7.53 1H,d H-3
2 δ 7.02 2H,d H-2´/ H-6´ (Ring B)
3 δ 6.97 2H,d H-3´/ H-5´ (Ring B)
4 δ 6.83 1H,d H-2
5 δ 6.68 1H,d H-2 (Ring A)
6 δ 6.66 1H,d H-6 (Ring A)
7 δ 6.56 1H,d H-5 (Ring A)
8 δ 3.28 2H,t H-1´ ́
9 δ 2.79 2H,t H-2´´
10 δ 2.19 1H,m -CH(isopropyl)
11 δ 1.00 3H,d CH3
12 δ 0.96 3H,d CH3
Table 3.6.3: Data of 13C NMR chemical shift of 3-(3, 4-dihydroxyphenyl)-N-[2-(4-isopropyl phenyl)-ethyl]acrylamide of Piper nigrum L.
S.no Benzene Ring A
13C NMR chemical
shift
Benzene Ring B
13C NMR chemical
shift
Other then ring
13C NMR chemical
shift
1 C-1 128.8 C-1´ 132.2 C-1 166.4 2 C-2 114..7 C-2´ 129.5 C-2 119.7 3 C-3 144.2 C-3´ 115.4 C-3 142.1 4 C-4 143.5 C-4´ 138.1 C-1´´ 37.3 5 C-5 118.0 C-5 ́ 115.4 C-2´ ́ 45.2 6 C-6 120.3 C-6 ́ 129.5 CH 31.6 7 - - - - 2 x CH3 24.3
Table 3.7.1: Data of IR and Mass spectrum of Trachyspermum ammi L.
1 Anal. Calcd C: 84.51; H: 11.74 2 for C30H50O C: 84.53: H:11.71 3 IR. (KBr) λmax 3420, 1630, 887 cm-1 4 Mass Spectrum
m/z426[H+]C30H50O 411,408,393, 218, 208, 203, 193, 188
Table 3.7.2: Data of 1H NMR chemical shift of Olean-12-ene-3β-ol of
Trachyspermum ammi L.
S.No Singlet Proton Assigned Of the proton 1 δ 5.24 1H, m H-12 δ 1.97-1.50 4H 2 δ 3.44 1H, m H-3 δ 1.49-1.31 10
H 3 δ 3.22 1H, m H-18 δ 1.29-1.15 8H 4 δ1.25-0.95 24H, s 8 x CH3
Table 3.7.3: Data of 13C NMR chemical shift of Olean-12-ene-3β-ol of
Trachyspermum ammi L. Carbon atom 13C NMR chemical
shift Carbon atom 13C NMR
chemical shift C-1 31.0 C-16 36.5 C-2 25.6 C-17 37.2 C-3 79.8 C-18 40.5 C-4 34.9 C-19 42.1 C-5 49.2 C-20 30.1 C-6 18.4 C-21 40.1 C-7 29.2 C-22 35.6 C-8 39.6 C-23 26.1 C-9 48.6 C-24 18.5 C-10 32.3 C-25 21.2 C-11 24.2 C-26 18.5 C-12 126.0 C-27 18.1 C-13 141.3 C-28 22.4 C-14 46.8 C-29 29.2 C-15 29.5 C-30 24.1
Fig. 3.9: 1H NMR chemical shift of 3β- hydroxy-olean-12 (13)-ene-28-oic acid of Nigella sativa L.
Fig. 3.10: 1H NMR chemical shift of 3-(3, 4-dihydroxyphenyl)-N-[2-(4-isopropylphenyl)-ethyl] acrylamide of Piper nigrum L..
Fig.3.11: 1H NMR chemical shift of 4-(2-propenyl) phenyl isobutyrate of
pimpinella anisum L.
Fig.3.12: 1H NMR chemical shift of Olean-12-ene-3β-ol of Trachyspermum ammi
L.
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