CHAPTER III ISOLATION AND CHARACTERIZATION OF …shodhganga.inflibnet.ac.in/bitstream/10603/62592/10/10_chapter 3.p… · or flavanones can form complex with anthocyanins (co –

96

CHAPTER III

ISOLATION AND CHARACTERIZATION OF FLAVONOIDS

Flavonoids are ubiquitous polyphenolic secondary metabolites in all the

plants. Flavonoids exhibit different biological functions that allow interactions

between plants and their environment; they are involved in the plant – pathogen

interaction, plant – plant interaction and plant – insect interaction [Treutter, 2005].

In the flowers, the flavonoids can act as a visual signal to attract pollinating animals.

Besides anthocyanins, responsible for red or purple colourations of petals, flavonols

or flavanones can form complex with anthocyanins (co – pigmentation) to give

strong blue colouration [Koes et al., 1994].

The photo protection from sunlight (UV) is a predominant role of flavonoids:

the epidermal flavonoids are strongly UV – absorbing and protect the internal tissues

of leaves and stems. Moreover flavonoids are potent scavengers of reactive oxygen

species and thus prevent peroxidation of lipids [Treutter, 2005]. Flavonoids have

received much attention due to their potential pharmacological properties. They have

been recognized to exert anti – allergenic, anti – artherogenic, anti – inflammatory,

antimicrobial, anti – thrombotic, antioxidant, cardioprotective and vasodilatory

effects [Manach et al., 2005].

The present investigation is focused at isolation of bioactive, novel

flavonoids from five plant species described in Chapter – II by following suitable

procedures in each case and its due characterization by interpretation of Rf, UV, 1H

and 13

C-NMR, FTIR and Mass data.

a. Sphaeranthus indicus Linn:

Materials and Methods:

Chemicals:

All the chemicals and reagents used were of analytical grade obtained from

S.D. Fine Chemicals Ltd., Mumbai, India.

97

Extraction and isolation:

The flower petals of S. indicus of about 1 kg were extracted with 85%

methanol (5 X 500 ml) under reflux. The alcoholic extract was concentrated in

vacuo and the aqueous concentrate was successively fractionated with petroleum

ether (60-80° C) (4 X 250 ml), peroxide – free diethyl ether (3 X 250 ml) and ethyl

acetate (4 X 250 ml). The petroleum ether and diethyl ether extracts yielded no

isolable crystalline solid and could not be studied further. The residue from ethyl

acetate fraction was taken up in a small quantity of acetone and left in an ice-chest

for a few days. The yellow solid (1) that separated was filtered and recrystallised

from methanol to give pale yellow micro crystalline solid (yield 0.1%).

Acid hydrolysis of the glycoside:

The glycoside (0.05 mg ≈ 0.2 m mol) dissolved in hot methanol (2 ml, 50%)

was hydrolysed with 5% H2SO4 at 100° C for about 2 hours. The excess alcohol was

distilled off in vacuo and the resulting aqueous solution was extracted with diethyl

ether. The aqueous filtrate was neutralized with BaCO3. An aliquot of this was

cautiously neutralized with NaHCO3 and the sugar was estimated quantitatively by

Folin-Wu micro method (Oser, 1965).

Paper chromatography - Rf values

Table III - 1

Rf (X 100) values of the constituents of flower petals of S. indicus

(Whatman No.1, Ascending, 30 ± 2°C)

Compound 1 a b c d e f g h

Glycoside ( G1 ) 38 41 49 57 69 55 73 65

Aglycone from (G1) after complete

hydrolysis

15 45 60 65 70 95 67 70

Solvent key: a – H2O, b – 5% aq.HOAc, c – 15% aq.HOAc, d – 30% aq.HOAc, e –

60% aq.HOAc, f – n – BuOH: HOAc: H2O = 4:1:5 (upper phase), g – Water

saturated with phenol and h – HOAc: Conc. HCl : H2O = 30:3:10.

Spray reagent: A mixture of 1% alcoholic solution of sodium borohydride and

AlCl3.

98

Table III - 2

Rf (X 100) values of the sugar from the Glycoside (G1) from S. indicus

(Whatman No.1, Ascending, 30 ± 2° C)

Compound 1 f g h i j

Sugar from G1 Glucose 18 38 38 - 24

Glucose (authentic) 17 38 37 - 24

Solvent key: i – EtOAc: Pyridine: H2O = 10:4:3, j – n – BuOH: Benzene: Pyridine:

H2O = 5:1:3:3.

Spray reagent: Aniline hydrogen phthalate

Table III - 3

13

C-NMR data signal assignments for the Glycoside (1) from Sphaeranthus

indicus

Compound 1

C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10

76.9 47.2 194.6 115.6 121.2 115.6 121.2 165.5 114.6

Compound 1

C-1' C-2' C-3' C-4' C-5' C-6'

129.0 125.3 148.4 145.5 114.6 125.3

Compound 1

C-1'' C-2'' C-3'' C-4'' C-5'' C-6'' C-5-CH3 C-7-CH3

96.0 70.8 76.8 70.2 73.3 61.3 17.7 18.9

99

Fig. III. 1. 1H-NMR Spectrum (DMSO-d6) of Compound 1

100

Fig. III. 2. 13

C-NMR Spectrum (DMSO-d6) of Compound 1

101

Fig. III. 3. FTIR Spectrum of the compound 1

102

Inte

nsi

ty

(cp

s)

Fig. III. 4. Mass Spectrum of compound 1

103

x

Results and Discussion

Compound (1) m.p. 278-80o

C. It developed green colour with alc. Fe3+

,

answered cyanidin test but not Wilson’s boric acid test [Wilson, 1939]. It responded

to Gibb’s [King et al., 1957] and Molisch’s but did not answer Hörhammer – Hansel

test. It developed an intense cherry – red colour when treated with Mg – HCl. It

MeOH

appeared fluorescent green when viewed under UV light. It had λmax 221, 290,

331; +NaOMe 273, 278, 382; +AlCl3 213, 290, 346; +AlCl3+HCl 216, 290, 326;

+NaOAc 213, 268, 322; +NaOAc+H3PO3 263, 290, 358 nm. 1H-NMR (400 MHz,

DMSO–d6, TMS) 7.0 (m, H – 6), 7.5 (q, H – 8), 6.8 (m, H – 2' and H – 5'), 6.4 (q, H

– 6'), 5.0 (q, H – 2), 2.5 (s, H – 3), 4.2 (d, H – 1'') 3.8 – 4.0 (other sugar protons), 1.8

– 1.9 ppm (m, Ar CH3 at C – 5 and C – 7), 13

C – NMR (75 MHz, DMSO-d6, TMS)

(Table III – 3) 115.6 (C – 5 and C – 7) 121.2 (C – 6 and C – 8), 165.5 (C – 9), 114.6

(C – 10,C – 5'), 129.0 (C – 1'), 125.3 (C – 2' and C – 6') 148.4 (C – 3'), 145.5 (C –

4'), 76.9 (C – 2), 47.2 (C – 3) 194.6 (C – 4), 17.7 (CH3 at C – 5), 18.9 (CH3 at C –

7), 96.0 (C – 1''),70.8 (C – 2''), 76.8 (C – 3''), 70.2 (C – 4''), 73.3 (C – 5''), 61.3 (C –

6''). FTIR υma KBr

cm-1

: 3419 (OH), 1720 (C=O). Its Rf values are presented in Table

III – 1. The molecular weight was calculated from the molecular formula, C23H26O10

(Molecular weight 462.4) based on mass spectroscopic study.The 1H,

13C-NMR,

FTIR and mass spectra of the glycoside are appended (Figs. III – 1, III – 2, III – 3

and III – 4).

The quantitative estimation of sugar by Folin – Wu Micromethod was in

agreement of a monoside. The concentrated filtrate indicated the presence of glucose

on PC using aniline hydrogen phthalate as spray reagent (Table III – 2). The residue

from diethyl ether fraction was characterized as aglycone of compound (1) by Rf

data (Table III – 1).

Compound (1) is characterized as 5, 7 – dimethyl – 3' – hydroxy flavanone –

4' – O – glucoside.

b. Zanthoxyleum tetraspermum Extraction and isolation:

The chipped woodhearts were washed with tap water followed by distilled

water, shade dried and powdered. The powdered woodhearts were subjected to

104

successive extraction with light petrol (60-80º C), peroxide free diethyl ether and

ethyl acetate using soxhlet extractor. The extracts were dried in vacuum pump at

40º C. The residue from ethyl acetate fraction was taken up in acetone and left in an

ice chest for two days when a pale yellow solid separated. It came out as pale yellow

plates compound (2) on recrystallisation from hot water. The dried crude extracts of

light petrol and diethyl ether were stored in freezer at 0º C. Light petrol and diethyl

ether extracts did not yield any isolable solid.

Compound (2) was characterised by Paper Chromatography (PC), UV, 1H

and 13

C – NMR, FTIR and mass spectral studies.


Table III - 4

Rf (X 100) values of the Constituents of the woodhearts of Z. tetraspermum



Glycoside ( G2 ) 36 60 68 70 82 64 51 88


hydrolysis

- - 05 - 49 93 67 87

Table III - 5

Rf (X 100) values of the sugar from the Glycoside (G2) from Z.tetraspermum


Compound 2 f g h I j



105

Table III - 6

13C-NMR data signal assignments for the Glycoside (2) from Z. tetraspermum

Compound

2

C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10

148.01 139.76 174.00 160.30 111.82 161.94 95.43 154.29 106.62

Compound 2

C-1' C-2' C-3' C-4' C-5' C-6'

119.17 129.00 114.82 155.99 114.82 129.00

Compound

2

C-1'' C-2'' C-3'' C-4'' C-5'' C-6'' C-5-

OCH3

C-7-

OCH3

100.65 73.80 76.81 70.31 75.69 63.11 55.69 56.40

Prenyl group

106

Fig. III. 5. 1H-NMR Spectrum (DMSO-d6) of compound 2

107

Fig. III. 6. 13

C-NMR spectrum (DMSO) of compound 2 (Expanded)

108

Fig. III. 7. 13

C-NMR spectrum (DMSO-d6) of compound 2

109

Fig. III. 8. FTIR Spectrum of compound 2

110

Inte

nsi

ty

(cp

s)

Fig. III. 9. Mass Spectrum of Compound 2

111

max

Result and Discussion

Compound (2) crystallised from ethyl acetae fraction and recrystallised from

acetone, came out as pale yellow plates (1g), m.p. 178-80º C. λMeOH

266, 285, 323;

+NaOMe: 262, 282; +AlCl3: 276,285, 327; + AlCl3 + HCl : 276, 282, 326; + NaOAc:

281, 322; + NaOAc + H3BO3: 278, 282, 324. 1H NMR (400MHz, Cd3Od) δ 6.80

(1H, s, H – 6), 7.00 (2H, d, J= 8.5 Hz, H-3' and H-5') 7.60 (2H, d, J=8.5 Hz, H – 2' and

H – 6'), 5.0 (1H, d, J=7 Hz, H – 1''). The vinylic methine proton of prenyl residue

appeared at δ 5.40 ppm (t, J = 4 Hz). The resonance of allylic methylene protons

appeared at1.80 ppm. Two methyl groups of prenyl residue appear at δ 1.20 and 1.40

ppm respectively. 13

C NMR (75.43 MHz, DMSO – d6): 148.01 (C – 2), 139.76 (C –

3), 174.00 (C – 4), 160.30 (C – 5), 111.82 (C – 6), 161.94 (C – 7), 95.43 (C – 8),

154.29 (C – 9), 106.62 (C – 10), 119.17 (C – 1'), 129.00 (C – 2'), 114.82 (C – 3'),

155.99 (C – 4'), 114.82 (C – 5'), 129.00 (C – 6'), 100.65 (C – 1''), 73.80 (C – 2''),

76.81 (C – 3''), 70.31 (C – 4''), 75.69 (C – 5''), 63.11 (C – 6''), 55.69 (5 – OCH3), 56.40

(7 – O – CH3). IR (νmax) Cm-1

3372, 2925, 2858, 1713, 1454, 1274, 825. From mass

spectral datas, the molecular weight of the compound is 544.5 (Molecular formula:

C28H32O11). Its Rf values are shown in table (III-4). The 1H and

13C-NMR, FTIR and

mass spectra are appended (Figs. III – 5, III – 6, III – 7, III – 8 and III – 9).

UV and IR spectra and a positive Molisch test indicated compound (2) to be

a flavonol glycoside with substitution at 4' –hydroxyl [Markham, 1982]. Hydrolysis

of compound (2) with mineral acid gave its aglycone and D – glucose. The β linkage

of the glucose moiety to 4' – hydroxyl group was evident from the large coupling

constant of H – 1'' signal in 1H NMR of compound (2) [Mabry et al, 1970].

The upfield shift by 3.21 ppm of C – 4' in 13

C NMR of compound (2) as

compared to the signals of authentic kaempferol (δ 159.20) further supported

glycosylation at C – 4' [Cussans and Huckerby, 1975]. The prenyl moiety at C – 6

and methoxy groups at C – 5 and C – 7 exhibited due resonances in 13

C – NMR. On

the basis of the above, compound (2) was characterised as 6 – prenyl – 5, 7 – di – O

– methyl Kaempferol 4' – O – glucoside. The hydrolysed aglycone of compound (2)

was characterised as 6 – prenyl – 5, 7 – di – O – methyl Kaempferol.

112

max

c. Cuscuta reflexa

Extraction and Isolation:

The fresh light yellowish plant (whole part) (1 kg) of Cuscuta reflexa

(parasite) collected from Kandaswami Kandar’s College, Velur in Namakkal District

during November were extracted with 85% methanol (5 X 500 ml) under reflux. The

combined alcoholic extract was concentrated in vacuo. The combined alcoholic

extract was successively fractionated with light petrol (60 – 80° C) (4 X 300 ml),

peroxide – free diethyl ether (3 X 300 ml) and ethyl acetate (3 X 250 ml) and the

respective fractions were collected and studied separately. The petrol and ether

fractions did not yield any isolable solid and were discarded.

Ethyl acetate fraction: flavonol glycoside 6 – prenyl kaempferide – 3 – O –

arabinosyl neohesperidoside.

The residue from ethyl acetate fraction was taken up in acetone and left in an

ice chest for two days. A yellow solid compound (3) that separated was filtered and

studied. It was recrystallised from methanol when it came out as yellow needles,

m.p. 177 – 79° C (yield 0.07%). It developed a green colour with alc. Fe3+

and a

pink colour with Mg – HCl. It appeared purple under UV that turned yellow on

exposure to NH3. It responded to Wilson’s boric acid, Molisch’s and Gibb’s tests but

did not answer the Hörhammer – Hansel test. It had λMeOH

266, 284 sh, 341;

+NaOMe 266, 289 sh, 385; +AlCl3 272, 298, 352, 392; +( AlCl3 – HCl) 272, 298,

353, 392; +NaOAc 278, 285 sh, 349: +(NaOAc – H3BO3) 280 sh, 336, 344 nm. The

molecular weight was calculated from the molecular formula C38H48O20 (Molecular

weight: 824) based on mass spectral study. Its Rfvalues are presented in table III – 7.

The 1H and

13C – NMR, FTIR and mass spectra are appended (Figs. III – 10, III –

11, III – 12 and III – 13).

Acid hydrolysis of the glycoside:

The glycoside (3) (0.05 g ≈ 0.2 m mol) dissolved in hot MeOH (2 ml, 50%)

was hydrolysed with H2SO4 (5%) at 100° C for about 2 hrs and the hydrolytic

products were identified as described below.

113

Identification of the aglycone: (Flavonol: Kaempferide)

The aglycone on recrystallisation from methanol appeared as yellow leaflets,

MeOH

m.p. 227 – 29° C (yield 0.02%). It had λmax 253 sh, 267, 299 sh, 320, 367;

NaOMe 280, 323 sh, 411; +AlCl3 (with and without HCl) 256, 270, 305 sh, 347,

422; +NaOAc 259 sh, 280, 301 sh, 384 and +(NaOAc-H3BO3) 255sh, 290 sh, 349,

367 nm. It was soluble in organic solvents but insoluble in water. It developed a

reddish orange colour with Mg – HCl and yellow colour with NaOH. It appeared

pale yellow under UV and showed greenish yellow fluorescence when fumed with

NH3. It responded to Wilson’s boric acid, Horhammer – Hansel and Gibb’s tests, but

did not answer the Molisch’s test. It afforded a triacetate, m.p. 193-95° C and a

tribenzoate, m.p. 178 – 79° C. It had Rf values as depicted in table III-7. It was

identified as 6- prenyl kaempferide by Co – and mixed – PC and m.m.p. with an

authentic sample of the same from Dillenia indica [Pavanasivam and Sultanbawa,

1975].

Identification of the sugar: (glucose, rhamnose and arabinose)

The aqueous solution from the above hydrolysate was neutralized with

BaCO3 and filtered. The concentrated filtrate indicated the presence of glucose,

rhamnose and arabinose on PC using aniline hydrogen phthalate spray reagent

(Table III – 8). The identity of the sugars was confirmed by Co – PC with authentic

samples of glucose, rhamnose and arabinose. A quantitative analysis revealed the

aglycone: sugar ratio to be 1:3. The glycoside was subjected to partial hydrolysis

with 10% formic acid in cyclohexanol [Harborne, 1964 and Fox et al., 1953]. The

resulting solution was extracted with ethyl acetate and subjected to PC. The products

were identified as 6 – prenyl kaempferide 3 – O – neohesperidoside and arabinose

which indicated that the terminal sugar was arabinose.

114


Table III - 7

Rf (X 100) values of the Constituents of whole plant parts of Cuscutta reflexa



Glycoside ( G3 ) 42 58 62 67 78 56 30 76

Glycoside resulting from partial

hydrolysis of (G3)

41 55 55 64 75 88 15 86


hydrolysis

08 08 14 17 11 88 15 70

The paper chromatogram was fumed with NH3 vapours.

Table III - 8

Rf (X 100) values of the sugar from the Glycoside (G3) from C.reflexa





Rhamnose 35 57 58 54 -

Rhamnose (authentic) 34 58 58 55 -

Arabinose 19 44 - - 29

Arabinose (authentic) 18 43 - - 29

115

Table III - 9

13C-NMR data signal assignments for the Glycoside (3) from Cuscutta reflexa

Compound 3

C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10

156.3 131.4 177.4 161.2 116.5 164.2 93.5 160.0 116.2

Compound 3

C-1' C-2' C-3' C-4' C-5' C-6' C-4'-OCH3

121.9 129.7 115.9 148.4 115.2 130.8 60.0

Compound 3

C-1'' C-2'' C-3'' C-4'' C-5'' C-6''

101.9 77.5 76.5 73.2 75.8 60.9

Compound 3

C-1''' C-2''' C-3''' C-4''' C-5''' C-6'''

103.9 72.3 74.8 71.2 67.9 22.0

Compound 3

C-1'''' C-2'''' C-3'''' C-4'''' C-5''''

100.9 74.1 70.5 69.9 60.8

Prenyl group

121.02 17.82

127.18

25.04 24.06

116


117

Fig. III.11. 13


118


119

Inte

nsi

ty (

cps)

Fig. III. 13. Mass spectrum of compound 3

120

Result and Discussion

The plants of Cuscutta reflexa have been found to contain 6 – prenyl

kaempferide – 3 – O – arabinosyl neohesperidoside.

The principal absorption maxima of the glycoside (3) and its aglycone are

respectively 341 nm (band I), 266 nm (band II) and 367 nm (band I) 267 nm (band II),

indicating a flavonol skeleton in both [Markham, 1982]. A comparison of band I

absorption of the glycoside and that of the aglycone reveals that there may be 3 –

glycosylation in the flavonol [Harborne et al., 1975]. This is also supported by the fact

that the glycoside did not respond to the Horhammer – Hansel test whereas the aglycone

did. The glycoside could be hydrolysed (5% H2SO4 100°, 2h) to 6-prenyl kaempferide,

glucose, rhamnose and arabinose. The presence of a free – OH at C – 5 in the glycoside

and the aglycone is evident from its positive response to Wilson’s boric acid test.

The same observation also stems from the fact that a bathochromic shift of 51

nm and 55 nm were observed in the glycoside and aglycone respectively in the AlCl3 –

HCl spectra [Jurd, 1962]. A bathochromic shift of 44 nm each in the glycoside and

aglycone in NaOMe spectra is suggestive of oxygenation at C – 4' in both [Harborne,

1988]. That no additional shift in AlCl3 spectrum as compared to AlCl3 – HCl spectrum

of the glycoside as well as that of aglycone is an indicative of the absence of catechol

type of substitution in B ring [Voirin, 1983]. This is also supported by the absence of

any characteristic shift (band I) is NaOAc – H3BO3 spectra of both the glycoside and

aglycone [Jurd et al., 1972].

The presence of a – OH group at C – 7 both in glycoside and aglycone could be

inferred from a bathochromic shift of 12 nm and 13 nm on the addition of NaOAc in

band II [Harborne et al., 1975].

In the 1H – NMR spectrum (400 MHz, DMSO – d6, TMS) (Fig. III – 10), the A

ring protons at C – 6 and C – 8 appear separately as doublets at δ 6.25 ppm (J = 2.5 Hz)

and 6.35 ppm (J=2.5Hz) [Harborne et al., 1975]. In the B ring, the protons at C – 2', C –

3', C – 5' and C – 6' due to free rotation of phenyl ring appear as two pairs of ortho

121

coupled doublets at δ 8.10 ppm (J = 8.5 Hz) and 6.80 ppm (J = 8.5 Hz). H – 3', H – 5'

doublet occurs upfield from H – 2', H – 6' doublet due to shielding effect of oxygenation

at C – 4' [Harborne et al., 1975]. The signals at δ 12.60 ppm and δ 11.00 ppm are

indicative of – OH protons at C – 5 and C – 7 respectively as evidenced from the

corresponding data culled out from literature [Mabry et al., 1970].

The β linkage of the glucose moiety to 3 – OH is evident from H – 1'' signal

appearing as a complex multiplet at δ 5.15 ppm [Mabry et al., 1970]. The H – 1''' of

rhamnose resonates at δ 4.50 ppm (d, J=2.0Hz). The appearance of methyl protons of

rhamnose at δ 1.10 ppm as a broad signal clearly reveals the presence of

neohesperidoside H – 1'''' of arabinose resonates at δ 4.90 ppm [Roster et al., 1965]. The

rest of the sugar protons appear in the range of δ 3.60 – 3.80 ppm.

The vinylic methine proton of prenyl residue appears at δ 5.40 ppm (t, J = 4 Hz).

The resonance of allylic methylene protons of the same residue appears at δ 2.50ppm.

Two methyl groups of prenyl group appear at δ 1.75 and 1.85 ppm respectively. The 3

proton signal at 3.60 ppm is assigned to – OCH3 group at C – 4' [Favid, 1918;

Rodrignez et al., 1972].

The structure of the glycoside could also be confirmed by the 13

C-NMR (75.43

MHz, DMSO – d6, TMS) spectrum (Fig. III – 11). The signal positions and their

complete assignment to different carbons are given in table III – 9. The C – 6 signal

appears at δ 116.5 ppm due to the attachment of prenyl substituent to it. As a result of

glycosylation the signal at C-3 is shifted upfield and appears at δ 131.4 ppm [Markham

et al., 1978]. C – 1'' of glucose appears at δ 101.90 ppm, C – 1''' of rhamnose at δ 103.90

ppm and C – 1'''' of arabinose at δ 100.90 ppm. The appearance of C – 6''' signals at δ

22.0 ppm (not at δ 17.40 ppm) and the signal of C – 6'' at δ 60.90 ppm (not at δ 68.00

ppm) clearly shows neohesperidose unit of the sugar moiety. Viz the linkage of glu →

rha is 1→ 2 [Ternai and Markham, 1976]. The appearance of the other arabinosyl

carbons in the range of δ 69.90 – 74.10 ppm (not between δ 82.00 and 61.00 ppm)

indicates that it is in the pyranose form [Rosenthal and Fendler, 1976]. The methoxyl

carbon at C – 4' resonates at δ 60.00 ppm.

122

From the foregoing evidences the glycoside from ethyl acetate fraction of

Cuscuta reflexa could be characterized as 6 – prenyl kaempferide – 3 – O – arabinosyl

neohesperidoside. This is a new record of a rare trioside from C.reflexa.

d. Citrullus colocynthis

Extraction and fractionation:

Fruits of Citrullus colocynthis (1 kg) were collected from the banks of river

Cauvery in Kutchipalayam of Namakkal district during September 2009. They were

extracted with boiling ethanol (85 %, 4 X 500 ml) and the combined extract was

concentrated in vacuo. The aqueous concentrate was successively fractionated with

petroleum ether (60 – 80° C) (4 X 250 ml), peroxide – free diethyl ether (2 X 250 ml)

and ethyl acetate (5 X 500 ml). The petroleum ether fraction was not taken up for study

as there was no indication of realizing any crystalline material from the same.

Isolation and idendification:

The diethyl ather fraction was concentrated in vacuo and left in an ice-chest for

a week. The yellow solid that separated was filtered and studied. It came out as pale

creamy yellow leaflets, m.p. 242-244°C (yield 0.02 %) on recrystallisation from

max

methanol. It had λMeOH 290, 330; +NaOMe 325, 332; +AlCl3 315, 330; +(AlCl3-HCl)

315, 332; +NaOAc 325, 330 and +(NaOAc – H3BO3) 290, 330 nm. It was identified as

hesperetin on the basis of colour rections, co – and mixed – Paper Chromatography and

mixed melting point with an authentic sample of hesperetin [Sulochana et al., 1989].

Ethyl acetate fraction: flavanone glycoside: hesperidin

The residue from ethyl acetate fraction was dissolved in acetone and the mixture

was left in an ice-chest for a week. A yellow solid compound (4) m.p. 244 – 246° C

(yield 0.06 %), that separated on recrystallisation from methanol came out as yellow

crystals. It developed a cherry – red colour when treated with Mg – HCl, a green colour

with alcoholic Fe3+ and fluoresced yellow under UV and UV / NH3. It responded to

Gibb’s and Molisch’s tests, but did not answer Horhammer – Hansel test. It answered

max

cyanidin test, but not the Wilson’s boric acid test. It had λMeOH 285, 328; +NaOMe

123

240, 284, 316, 354; +AlCl3 310, 385; + (AlCl3 – HCl) 304, 377; +NaOAc 286, 330 and

+(NaOAc – H3BO3) 282, 324 nm. The molecular weight was calculated as 610

(Molecular formula: C28H34O15) from mass spectra. Its Rf values are presented in table

III – 10. The 1H,

13C-NMR, FTIR and mass spectra of the glycoside are appended (Figs.

III – 14, III – 15, III – 16 and III – 17).

Hydrolysis of the glycoside:

To a solution of the glycoside (4) (100 mg) in hot aq. methanol (10 ml, 50 %) an

equal volume of 5% H2SO4 was added and the mixture was gently refluxed at 100° C

for 2 hour. The excess alcohol was distilled off in vacuo and the resulting aq.solution

was extracted with diethyl ether.

Identification of the aglycone: (hesperetin)

The residue from the diethyl ether fraction was taken up in acetone and left

under chilled conditions for a few days when a creamy yellow solid was obtained. Its

colour reactions, chromatographic behaviour and UV spectral data were all similar to

those described for the free aglycone mentioned earlier.

Identification of the sugar: (glucose and rhamnose)

The filtrate after the removal of aglycone was neutralized with BaCO3. The

concentrated filtrate when examined by paper chromatography gave Rf values

corresponding to those of glucose and rhamnose (Table III – 11). The running

properties of the glycoside were also in favour of a bioside. The identity of the sugars

was confirmed by comparison with authentic samples of glucose and rhamnose.

Enzymatic hydrolysis with pectinase [Markham, 1982]

To 5 mg of the glycoside (4) in distilled water (0.5 ml) about 6 flakes of pectinase

(Koch – light, from Aspergillus niger) were added. The reaction mixture was left overnight.

It was then concentrated. The residue was dissolved in water and chromatographed (PC) in

15 % HOAc. The completion of hydrolysis could be inferred from the Rf values (different

from those of the glycoside) which agreed with those of hesperetin. Thus the linkage of

sugarwas confirmed to be β – in glucose and α – in rhamnose.

124

Partial hydrolysis of the glycoside [Harborne, 1964; Fox et al., 1953]

The glycoside was subjected to partial hydrolysis by treatment with 10 % formic

acid in cyclohexanol and the resulting solution was extracted with ethylacetate and

subjected to PC. The Rf values of the ethyl acetate fraction agreed with those of

hesperetin 7 – O – glucoside. The Rf values are indicated in table III – 10. It derived to

conclude that glucose is directly linked to the aglycone moiety.


Table III-10

Rf (X 100) values of the Constituents of C. colocynthis

(Whatmann No.1, Ascending, 30 ± 2° C)


Glycoside ( G4 ) 26 47 63 69 72 64 92 60

Glycoside resulting from partial hydrolysis

of compound 4

05 14 25 44 68 76 90 64

Aglycone from compound 4 after complete

hydrolysis

11 49 61 67 73 90 68 64

Table III - 11

Rf (X 100) values of the sugar from the Glycoside (G4) from C. colocynthis





Rhamnose 34 58 58 55 -

Rhamnose (authentic) 34 58 58 55 -

125

Table III-12

13

C-NMR data signal assignments for the Glycoside (4) from C. colocynthis

Compound 4 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10

Glycoside (4) (δppm) 78.5 41.0 196.1 170.0 98.8 163.0 96.5 153.6 99.8

Hesperetin from

literature δppm

78.5 42.1 196.2 163.8 96.2 166.9 95.4 163.0 102.1

Compound 4 C-1' C-2' C-3' C-4' C-5' C-6' C of OCH3

Glycoside (4)

(δppm)

127.7 120.1 136.3 145.2 120.6 116.5 -

Hesperetin from

literature δppm

131.4 114.3 146.7 148.1 112.1 118.0 56.0

Compound 4 C-1'' C-2'' C-3'' C-4'' C-5'' C-6''

Glycoside (4) (δppm) 99.6 73.2 76.6 72.5 76.8 68.3

Rutinose from literature δppm 99.7 73.3 76.6 71.0 75.8 66.4

Compound 4 C-1''' C-2''' C-3''' C-4''' C-5''' C-6'''

Glycoside (4) (δppm) 100.7 71.1 69.4 72.7 69.3 17.7

Rutinose from literature δppm 100.9 70.6 69.9 72.4 68.6 17.4

126


127

Fig. III. 15. 13


128

Fig. III. 16. FTIR spectrum of compound 4

129

Inte

nsi

ty

(cp

s)

Fig. III. 17 Mass spectrum of compound 4

130


The fruits of citrullus have been found to contain hesperetin and its 7 – O –

rutinoside (hesperidin).

The free aglycone from the diethyl ether fraction and the aglycone obtained

after the hydrolysis of the glycoside could be characterized as hesperetin by λmax

diagnostic shifts, Rf values and colour reactions.

The UV spectrum of the glycoside (4) exhibited an inflection of low intensity at

328 nm (band I) and a peak at 285 nm (band II). Absence of any bathochromic shift

in band II in NaOAc spectrum of the glycoside and the bathochromic shift observed

in the corresponding spectrum of the aglycone suggest the site of glycosylation at C

– 7 [Harborne et al., 1975].

In the 1H-NMR spectrum of the glycoside (400MHz, CD3OD, TMS) (Fig. III

– 14), the signal at δ 5.83ppm corresponds to the proton at C – 6, whereas the proton

at C – 8 resonates at δ 6.12 ppm. The signal centered at δ 6.96 ppm integrates for 3

protons at C – 2', C – 5' and C – 6' [Harborne et al., 1975]. The proton at C – 2

appears at δ 4.92 ppm. Owing to spin – spin interaction with each other and with C –

2 proton, the two protons at C – 3 resonate at δ 2.80 ppm as a quartet [Mabry et al.,

1970]. The signal at δ 3.82 ppm integrates for 3 protons indicating a methoxyl group

[Favid, 1918 and Rodrignez et al., 1972]. H – 1'' of glucose and H – 1''' of rhamnose

appear at δ 4.54 ppm and 4.64 ppm respectively. The signal appearing in the range

of δ 0.90 – 1.10 ppm corresponds to C – 6''' protons (methyl protons) of rhamnose

and is clearly reminiscent of the presence of a rutinoside [Markham and Mabry,

1962]. Had it been a neohesperidoside where the linkage is 1→2, the corresponding

signal would have appeared at δ 1.10 – 1.30 ppm. The broad multiplet at δ 3.50 ppm

can be traced to the other protons of rhamnoglucosyl moiety.

The structure of the glycoside could also be confirmed by its 13

C – NMR

(100.61 MHz, DMSO-d6, TMS) (Fig. III – 15) spectrum. The signal positions and

their complete assignment to different carbons are given in table III – 12. A perusal

of literature and a comparison of the signal positions of the glycoside and aglycone

indicate that C – 7 is shifted upfield by 3.9 ppm and C – 6 and C – 8 are shifted

downfield by 2.6 ppm and 1.1 ppm respectively [Markham et al., 1978]. The signal

131

λMeOH

of C – 6''' of rhamnose at δ 17.7 ppm (not at δ 20.9 ppm) and that of C – 6'' of

glucose at 68.3 ppm (not at δ 60.9 ppm clearly show that the glycoside is a 7 – O –

rutinoside [Ternai and Markham, 1976].

On this basis, the glycoside from the ethyl acetate fraction was characterized

as hesperetin 7 – O – rutinoside (hesperidin).

d. Argemone Mexicana L.

Air dried leaves (1Kg) of Argemone Mexicana L. collected from Mohanur,

Namakkal District, Tamil Nadu, India during March – April 2009, were extracted

with 85% methanol (5 X 500 ml) under reflux. The combined alcoholic extract was

concentrated in vacuo. The aqueous concentrate was subjected to extraction with

light petrol (60-80º C) (4 X 500 ml). After evaporation of the solvent, the extract

was saponified with alcoholic KOH, to remove fatty material. This yields 10 g of

unsaponified matter.

A small quantity of unsaponifiable matter was dissolved in chloroform and

this solution was spotted on TLC plats. Then the TLC plates were run by specific

solvent system and were viewed individually under UV light. Through several pilot

experiments it was found that the compounds of unsaponifiable fraction were

separated by the solvent system of light petrol and chloroform 10:1. The

unsaponifiable fraction, 8g was subjected to column chromatography on silica gel

(60 – 120 mesh) with gradient elution using light petrol: chloroform. The fraction

which was homogeneous on TLC plate by using light petrol: chloroform (10:1) was

crystallized. The solid that separated was recrystallised from light petrol when it

came out as colourless needles, m.p. 165 – 67º C. It answered negative for usual

characteristic colour reactions of flavonoids, alkaloids and terpenoids. It had

max 234, 269, 330; +NaOMe 242, 269; +AlCl3 233, 269, 335; + (AlCl3 – HCl)

237, 270, 334; +NaOAc 231, 267, 335; +NaOAc – H3BO3 230, 268, 334 nm. The 1H

and 13

C – NMR are appended.

132

Table III-13

13

C-NMR data signal assignments for the A. Mexicana L.

Compound 5

C-1 C-2 C-3 C-4 C-5 C-6

137.4 108.3 126.8 132.9 145.3 123.4

Compound 5

C-1' C-2' C-3' C-4' C-5' C-6'

120.1 150.3 101.9 150.3 105.3 130.6

Compound 5

C-α C-β and C-β' C-5-OCH3 C-2'-OCH3 C-4'-OCH3

26.7 38.7 61.8 57.0 55.1

133

Fig. III. 5. 1H-NMR spectrum (DMSO-d6) of compound 5

134

Fig. III. 19. 13


135


136

Inte

nsi

ty

(cp

s)

Fig. III. 21. Mass spectrum of compound 5

137


Air dried leaves of A. Mexicana L.have been found to contain 5, 2', 4' – tri –

O – methyl chalcane.

The principal absorption maxima of the recrystallised light petrol extract are

330 nm (band I) and 264 nm (band II). With various shift reagents, no significant

shift to principal absorption maxima was observed.

In the 1H – NMR spectrum (400 MHz, DMSO – d6, TMS) (Fig. III – 18) the

two protons singlet at δ 2.15 ppm is assigned to methylene protons at C – α of

chalcane unit, whereas the equivalent four protons at β – carbons resonate as singlet

at δ 2.80 ppm. The three signals at δ 3.4, 4.15 and 4.95 ppm each integrating for 3

protons indicate the methoxy groups in the aromatic systems at C – 3' and C – 2' and

C – 5 respectively [Favid, 1918; Rodrignez et al., 1972]. The aromatic methine

proton resonances H – 2, H – 3, H – 4, H – 3', H – 5' and H – 6 appear between δ 6.0

– 9.9 ppm.

The structure of the glycoside could also be confirmed by 13

C – NMR (75.43

MHz, DMSO – d6, TMS) (Fig. III – 19). The signal positions and their complete

assignment to different carbons are given in table III – 13. C – α appear at δ 26.2

whereas the two C – β appears at δ 39.8 ppm. The methoxy carbons resonate at δ

55.1, 57.0 and 61.8 ppm [De Almeida et al., 1979]. The molecular weight of the

compound was calculated from the molecular formula C18H22O3 (Molecular weight

286) based on the mass spectroscopic study.

From the above evidences the compound isolated from A. mexicana could be

confirmed as 5, 2', 4' – tri – O – methyl chalcane. Compounds belonging to

chalcanoids are derived from 1, 3 – diaryl propane as the parent carbon skeleton.

Chalconids without hydroxyl group in C – 3 system are designated as chalcane. The

important feature of these compounds is the absence of carbonyl groups [Agrawal,

1989].

This is the first report of a chalcane from Argemone Mexicana L. leaves.

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