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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