New constituent from Argemone mexicana Linn.

Article abstract of DOI:10.14233/ajchem.2014.15450

The present paper reports the isolation and structure elucidation of a new compound A identified as 5,7,4′-trihydroxy-8-methoxy flavanone-4′- α-L-rhamno-pyranosyl-7-O-β-D-xylopyranosyl-(1→3) -O-β-D-galactopyranoside along with two known compounds B and C identified as chrysoeriol and tricin, respectively from the methanolic extract of the defatted seeds of the plant by various colour reactions, chemical degradation and spectral analysis.

Full text of DOI:10.14233/ajchem.2014.15450

Asian Journal of Chemistry; Vol. 26, No. 2 (2014), 472-474
http://dx.doi.org/10.14233/ajchem.2014.15450
New Constituent from Argemone mexicana Linn.
1
2
2,*
RAJ N. YADAVA , JAY D. SHARMA and ANUREKHA YADAV
1Natural Products Laboratory, Department of Chemistry, Dr. H.S. Gour University, Sagar-470 003 India
2Department of Criminology and Forensic Science, Dr. H.S. Gour University, Sagar-470 003 India
*Corresponding author: anu27_yadav@yahoo.co.in
Received: 8 March 2013;
Accepted: 11 September 2013;
Published online: 15 January 2014;
AJC-14569
The present paper reports the isolation and structure elucidation of a new compound A identified as 5,7,4'-trihydroxy-8-methoxy
flavanone-4'-α-L-rhamno-pyranosyl-7-O-β-D-xylopyranosyl-(1→3)-O-β-D-galactopyranoside along with two known compounds B and
C identified as chrysoeriol and tricin, respectively from the methanolic extract of the defatted seeds of the plant by various colour
reactions, chemical degradation and spectral analysis.
Keywords: Argemone mexicana Linn. Papaveraceae, Seeds, Chrysoeriol, Tricin.
solvent. Mass spectra were recorded on a Jeol SX-102 (FAB)
Mass spectrometer.
INTRODUCTION
Argemone mexicana Linn.1-4 belongs to Papaveraceae
family. It is commonly known as 'Bharbhand' or 'Shialkanta'
in Hindi. It is a native of Mexico which has run wild in India
and is now a troublesome weed. It grows in wasteland and is
abundantly found in Asia and Africa over a much extended
area. Its medicinal value includes usage of root and stems for
chronic skin diseases. Its juice or latex is recommended for
treatment of abnormal accumulation of liquid in cellular tissue.
Its seeds are also used as a laxative and for removal of mucous
secretions from bronchial tubes. Its seeds resemble black
mustard seeds and are therefore sometimes found mixed with
it and this mixing of argemone oil in mustard oil is probably
responsible for outbreaks of epidemic dropsy. Previous
workers5,6 have reported various constituents from this plant.
This paper deals with the isolation and structural elucidation
of compound A, identified as 5,7,4'-trihydroxy-8-methoxy
flavanone-4'-α-L-rhamnopyranosyl-7-O-β-D-xylopyranosyl-
(1→3)-O-β-D-galactopyranoside along with two known
compounds, B identified as chrysoeriol and compound C
identified as tricin from the methanolic extract of the defatted
seeds of the plant.
The seeds of Argemone mexicana Linn. were procured
from Sagar region and were taxonomically authenticated by
the Department of Botany, Dr. H.S. Gour University, Sagar. A
voucher specimen has been deposited in the Natural Products
Laboratory, Department of Chemistry, Dr. H.S. Gour University,
Sagar, India.
Extraction and isolation: Air-dried and powdered seeds
(4 kg) of the plant were extracted with petroleum ether (40-60
°C) in a Soxhlet apparatus for 4 days. The seeds were then
successively extracted with CHCl3, CH3COOC2H5, CH3COCH3
and CH3OH. The methanol soluble fraction of the plant was
concentrated under reduced pressure to give brown viscous mass.
It gave three spots on TLC examination using chloroform:
methanol:water (6:4:2) as solvent and I2 vapours as visualizing
agent indicating it to be mixture of three compounds A, B and
C. These were separated by column chromatography over
a silica gel column using CHCl3: MeOH (3:6) as eluent and
studied separately.
Compound A (Fig. 1): It was obtained as light brown
amorphous powder, m.p. 261-262 °C, m.f., C33H42O19, [M]+
m/z 742 (FABMS). Found C, 53.81 %; H, 5.62 %; Calcd. for
molecular formula C33H42O19: C, 53.37 %; H, 5.66 %. UV
(MeOH) λmax nm; 286, 328; IR (KBr, νmax, cm-1) 3276, 1630,
1596, 1312, 1249, 1182, 1158, 1078, 828; 1H NMR (300 MHz,
CDCl3): δ 5.23 (1H, dd, J = 2.3, 12.5 Hz, H-2), 2.81 (1H, dd,
J = 3.7, 16.8 Hz, H-3α), 3.18 (1H, dd, J = 11.8, 17.7 Hz, H-
3β), 5.69 (2H, s, H-6), 3.77 (3H, s, OMe-8), 7.24 (2H, d, J =
8.1 Hz, H-2', H-6'), 7.11 (2H, d, J = 8.2 Hz, H-3', H-5'), 12.23
EXPERIMENTAL
All the melting points were determined on a thermo-
electrical melting point apparatus and are uncorrected. The
IR spectra were recorded in KBr disc; 1H NMR and 13C NMR
spectra were recorded at 300 MHz on Bruker DRX 300 NMR
spectrometer using TMS as internal standard and CDCl3 as

Vol. 26, No. 2 (2014)
New Constituent from Argemone mexicana Linn. 473
CH3
OCH3
OH
O
OCH3
HO
O
O
O
O
O
HO
O
OH
OH
OH
HO
OH
OCH3
O
O
OH
HO
OH
O
OH
OH
Compound C
Fig. 3
Compound A
Fig. 1.
diethyl ether. The ethereal layer was washed with water and
the residue was chromatographed over silica gel using CHCl3:
MeOH as solvent to give aglycone A-1 which was identified
as 5, 7, 4'-trihydroxy-8-methoxy flavanone by comparison of
its spectral data with reported literature values. The aqueous
hydrolyzate was neutralized with BaCO3 and the BaSO4 was
filtered off. The filtrate was concentrated and subjected to paper
chromatography examination using n-butanol:acetic acid:
water n-BAW (4:1:5) as solvent system and the sugars so
obtained were identified as D-xylose (Rf 0.26), L-rhamnose
(Rf 0.36), D-galactose (Rf 0.15) by (Co-PC and Co-TLC).
Compound A-1 (Fig. 4): It is light yellow powder, m.p.
244-246 °C, m.f. C16H14O6; [M]+ 302 (FABMS); Found C,
65.55 %; H, 4.62 %; Calcd. for Molecular Formula C16H14O6,
C, 63.57 %; H, 4.64 % ; UV (MeOH) λmax nm; 284, 326; IR
(KBr, νmax, cm-1) 3272, 1632, 1394, 1310, 1252, 1186, 1160,
1054, 826; 1H NMR (300 MHz, CDCl3): δ 5.32 (1H, dd, J =
2.1, 12.3 Hz, H-2), 2.78 (1H, dd, J = 3.9, 16.5 Hz, H-3α), 3.21
(1H, dd, J = 11.6, 17.4 Hz, H-3β), 5.66 (2H, s, H-6), 3.81
(3H, s, OMe-8), 7.28 (2H, d, J = 8.4 Hz, H-2', H-6'), 7.19 (2H,
d, J = 8.6 Hz, H-3', H-5'), 12.29 (1H, s, 5-OH); 13C NMR
(300 MHz, CDCl3) δ 76.7 (C-2), 42.4 (C-3), 197.2 (C-4), 163.6
(C-5), 94.1 (C-6), 165.8 (C-7), 164.8 (C-8), 161.6 (C-9), 100.9
(C-10), 129.3 (C-1'), 128.8 (C-2'), 115.7 (C-3'), 158.1 (C-4'),
115.7 (C-5'), 128.8 (C-6').
(1H, s, 5-OH); 5.41 (1H, d, J = 7.2 Hz, H-1''), 4.58 (1H, dd, J =
2.9, 9.4 Hz, H-2''), 4.61 (1H, dd, J = 3.6, 9.3 Hz, H-3''), 4.66
(1H, d, J = 5.7 Hz, H-6'', CH3); 5.24 (1H, d, J = 2.1 Hz, H-1'''),
4.69 (1H, dd, J = 3.1, 9.8 Hz, H-2'''), 4.73 (1H, dd, J = 2.8, 9.7
Hz, H-3'''), 4.67 (1H, dd, J = 2.9, 10.7 Hz, H-4'''), 4.34 (1H,
m, H-5'''); 1.18 (3H, d, J = 6.1 Hz, H-6'''); 5.14 (1H, d, J = 6.4
Hz, H-1''''), 4.38 (1H, dd, J = 3.3, 10.7 Hz, H-2''''), 4.13 (1H,
dd, J = 3.5, 10.4 Hz, H-3''''), 4.27 (1H, m, H-4''''), 4.68 (2H, d,
J = 6.1 Hz, H-5''''); 13C NMR (300 MHz, CDCl3) δ 76.87
(C-2), 41.8 (C-3), 196.4 (C-4), 163.2 (C-5), 94.6 (C-6), 164.2
(C-7), 165.3 (C-8), 160.7 (C-9), 101.2 (C-10), 127.9 (C-1'),
129.3 (C-2'), 115.1 (C-3'), 157.6 (C-4'), 115.2 (C-5'), 129.1
(C-6'); 104.9 (C-1''), 84.82 (C-2''), 76.94 (C-3''), 71.03 (C-4''),
67.11 (C-5''), 17.71 (C-6', CH3); 105.2 (C-1'''), 74.54 (C-2'''), 74.87
(C-3'''), 70.12 (C-4'''), 75.88 (C-5'''), 60.36 (C-6'''); 102.56 (C-
1''''), 70.89 (C-2''''), 80.66 (C-3''''), 71.87 (C-4''''), 68.86 (C-5'''').
Compound B (Fig. 2): It was crystallized from MeOH to
give light yellow powder. m.p. 325-327 °C, m.f., C16H12O6 [M+]
300 (FABMS); Found C, 63.97 %; H, 3.96 %; Calcd. for
Molecular Formula C16H12O6, C, 64 %; H, 4 %, UV (MeOH)
λ
max nm; 266, 338; IR (KBr, νmax, cm-1) 3348, 1654, 1626, 1567,
1512, 1436, 1346, 1304, 1211, 1174, 1030, 996, 865, 838,
792, 760. 1H NMR (300 MHz CDCl3) δ 6.31 (1H, d, J = 2.3
Hz, H-6), 6.48 (1H, d, J = 2.4 Hz, H-8), 6,62 (1H, s, H-3),
7.12 (1H, d, J = 8.5 Hz, H-5), 7.54 (1H, dd, J = 2.0, 8.5 Hz,
H-6'), 7.71 (1H, d, J = 1.8 Hz, H-2''), 4.06 (1H, s, 4' OMe),
12.94 (1H, s, 5-OH).
OCH3
HO
O
OH
OCH3
HO
O
OH
OH
O
Compound A-1
Fig. 4
OH
O
Compound B
Permethylation of compound A: Compound A (20 mg)
was dissolved in DMF (30 mL) and treated with MeI (5 mL)
andAg2O (15 mg) in 150 mL round bottomed flask fitted with
condenser and refluxed for 2 days. The contents were filtered
and washed with DMF. The filtrate was concentrated under
reduced pressure and treated with CHCl3 (25 mL) and washed
with water. After removal of solvent a syrupy mass was found
which was hydrolyzed with 7 % H2SO4 (5 mL) to give methylated
aglycone, identified as 7,4'-dihydroxy, 5,8-di-methoxy flava-
none and methylated sugars. The aqueous hydrolyzate obtained
after the removal of aglycone was neutralized with BaCO3
and the BaSO4 was filtered off. The filtrate was concentrated
under reduced pressure and subjected to paper chromatography
on Whatmann filter paper No.1 using n-butanol:ethanol:water
(5:1:4) as solvent system and aniline hydrogen phthalate as
spraying agent. The methylated sugars were identified as 2,3,4-
Fig. 2.
Compound C (Fig. 3): It was obtained as light brownish
yellow powder crystallized from MeOH. m.p. 275-276 °C,
m.f., C17H14O7; [M+] 330 (FABMS); Found C, 61.79 %; H,
4.24 %; Calcd. for Molecular Formula C17H14O7, C, 61.81 %;
H 4.24 % ; UV (MeOH) λmax nm 272, 352, IR (KBr, νmax, cm-1)
1
3452, 1656, 1612, 1504, 1098, 1072, 1030; H NMR (300
MHz CDCl3) δ 7.42 (1H, s, H-2', H-6'), 6.76 (1H, s, H-3), 6.38
(1H, d, J = 1.8 Hz, H-8), 6.24 (1H, d, J = 2.2 Hz, H-6), 3.91
(3H, s, -OMe-3'), 3.90 (3H, s, OMe-5'), 12.83 (1H, s, 5-OH).
Acid hydrolysis of compoundA: CompoundA (500 mg)
was dissolved in ethanol (20 mL) and refluxed with 10 %
H2SO4 (10 mL) on water bath for 8 h. The contents were concen-
trated and allowed to cool and residue was extracted with

474 Yadava et al.
Asian J. Chem.
tri-O-methyl L-rhamnose, 2,4,6-tri-O-methyl D-galactose,
2,3,4-tri-O-methyl D-xylose.
galactose and 2,3,4-tri-O-methyl-D-xylose, confirming that
hydroxyl group at C-7 and C-4' position of aglycone were
involved in glycosidation.
Enzymatic hydrolysis of compound A: Compound A
(40 mg) was dissolved in MeOH (25 mL) and hydrolyzed with
equal volume of almond emulsin enzyme. The reaction mixture
was allowed to stay undisturbed at room temperature for 2 days
and filtered. The hydrolyzate was concentrated and subjected
to paper chromatography examination using n-BAW (4:1:5)
as solvent system and aniline hydrogen phthalate as a spraying
reagent which showed the presence of D-galactose (Rf 0.15)
and D-xylose (Rf 0.26). The proaglycone was dissolved in
MeOH (10 mL) and further hydrolyzed with equal volume of
enzyme takadiastase at room temperature which yielded
aglycone identified as 5,7,4'-trihydroxy-8-methoxy flavanone
and the sugar was identified as L-rhamnose Rf (0.36) by
Co-PC and Co-TLC.
Therefore it was concluded that C-1'' of L-rhamnose was
linked with C-4' position of aglycone and C-1''' of D-galactose
was linked to C-7 position of aglycone and also that C-3''' of
D-galactose is linked to C-1'''' of D-xylose. The inter glycosidic
linkage (1→3) was found between D-xylose and D-galactose.
Enzymatic hydrolysis16 of compound A with almond
emulsin enzyme liberated D-xylose, D-galactose and proaglycone
confirming the presence of β linkage between D-xylose and
D-galactose and also between D-galactose and proaglycone.
Proaglycone 5, 7, 4'-trihydroxy 8 methoxy flavanone -4'-α-L-
rhamnopyranosyl, on further hydrolysis with takadiastase
liberated L-rhamnose and aglycone, this confirmed the presence
of α-linkage between L-rhamnose and aglycone. Thus on the
basis of above evidence the structure of compound A was
established as 5,7,4'-trihydroxy-8-methoxy flavanone-4'-α-L-
rhamnopyranosyl-7-O-β-D-xylopyranosyl-(1→3)-O-β-D-
galactopyranoside.
RESULTS AND DISCUSSION
Compound A has m.p. 261-262 °C, m.f. C33H42O19 [M+]
742 (FABMS).
It gave positive Molisch7 and Shinoda8 test showing its
flavonoidal glycosidic nature. Its UV spectrum showed absor-
ption bands at 286 and 328 nm suggesting it to be a flavonoid.
Its IR spectrum showed bands at 3272 cm-1 for OH group and
at 1632 cm-1 for carbonyl group. In its 1H NMR spectra signals
at δ 7.24 (2H, d, J = 8.1 Hz) and at δ 7.11 (2H, d, J = 8.2 Hz)
were assigned to H-2', H-6' and H-3', H-5' respectively. Charac-
teristics peak were observed at δ 5.23 (1H, dd, J = 2.3, 12.5
Hz) for H-2 and at δ 2.81 (1H, dd, J = 3.7, 16.8 Hz) and δ 3.18
(1H, dd, J = 11.8, 17.7 Hz) for H-3α and H-3β respectively.
Three singlets at δ 5.69 for H-6 at δ 3.77 for OMe-8 and at δ
12.23 for OH group at H-5 were present. The anomeric
proton signals at δ 5.41 (1H, d, J = 7.2 Hz), δ 5.24 (1H, d, J =
2.1 Hz) and at δ 5.14 (1H, d, J = 6.4 Hz) were assigned to H-
1'' of L-rhamnose, H-1''' of D-galactose and H-1'''' of D-xy-
lose respectively. In 1H NMR spectrum coupling constant J =
2.1 Hz and J = 6.4 Hz value of anomeric proton of D-galac-
tose and D-xylose confir-med β configuration between galac-
tose and xylose and coupling constant J = 7.2 Hz value for
anomeric proton of L-rhamnose confirmed the α configura-
tion of L-rhamnose.
Compound B was analyzed for m.f., C16H12O6, m.p. 325-
327 °C [M+] 300 (FABMS) and was identified as Chrysoeriol by
comparison of its spectral data with reported literature values17.
Compound C was analyzed for m.f., C17H14O7, m.p. 275-
276 °C [M+] 330 (FABMS) and was identified as Tricin by
comparison of its spectral data with reported literature values18.
ACKNOWLEDGEMENTS
The authors are grateful to Head SAIF, Central Drug
Research Institute, Lucknow (U.P.) India for recording various
spectral data and also greatful to Head, Department of Chemistry,
Dr. H.S. Gour Central University, Sagar, India for providing
necessary laboratory facilities.
REFERENCES
1. R.N. Chopra, S.L. Nayar and I.C. Chopra, Glossary of Indian Medicinal
Plants, CSIR, New Delhi, 23, (1956).
2. J.C. Kurian, Plants that Heal, Oriental Watchman Publishing House
Pune, India, edn 1, pp. 177-178 (1995)
3. Col. H. Drury, Useful Plants of India with Notices of Their Value in
Commerce Medicine and the Arts, William H. Allen & Co. 13 Waterloo
place S.W., pp. 50-51 (1978).
4. The Wealth of India, Publication and Information Directorate, CSIR,
New-Delhi India, Vol. 14, 116, (1948).
Acid hydrolysis of compound A with 10 % ethanolic
H2SO4 gave aglycone A-1 m.p. 244-246 °C, m.f., C16H14O6
[M+] 302 (FABMS) identified as 5, 7, 4'-trihydroxy-8-methoxy
flavanone by comparison of its spectral data with reported lite-
rature values9-11. The aqueous hydrolysate obtained was
neutralized with BaCO3 and the BaSO4 was filtered off. The
filtrate was concentrated and subjected to paper chromato-
graphy12 showing the presence of L-rhamnose, D-xylose and
D-galactose. Quantitative estimation13 of sugars revealed that
all the three sugars were present in equimolecular ratio (1:1:1).
Periodate oxidation4 of compound A confirmed that all the
three sugars were present in pyranose form.
5. R.P. Rastogi and B.N. Mehrotra, Compendium of Indian Medicinal
Plants, PID, New Delhi, Vol. 3, 59, (2001).
6. S. Rajvaidya, B.P. Nagori, G.K. Singh, B.K. Dubey, P. Desai and S. Jain,
Int. J. Pharm. Sci. Res., 3, 2494 (2012).
7. F.G. Mann and B.C. Saunders, Practical Organic Chemistry, Orient
Longmann, edn 4, p. 367 (1999).
8. J. Shinoda, J. Pharm. Soc. Jpn., 48, 214 (1928).
9. G. Chen, C. Dai, T. Wang, C. Jiang, C. Han and X. Song, Arkivoc,
179 (2010).
10. Y.H. Kuo, S.M. Lee and J.S. Lai, J. Chin. Chem. Soc., 47, 241 (2000),
11. A. Hasan and N.N. Tahir, Turk. J. Chem., 29, 65 (2005).
12. E. Lederes and M. Lederer, Chromatography, Elsevier Publishing Com-
pany, New York, vol. 1, p. 247 (1947).
13. S.B. Mishra and V.K.M. Rao, J. Sci. Ind. Res., VC19, 173 (1960).
14. E.L. Hirst and J.K.N. Jone, J. Chem. Soc., 1659 (1949).
15. S. Hakomoni, J. Biochem., 55, 205 (1964).
The position of the sugar moieties in compound A were
determined by permethylation14,15 followed by acid hydrolysis,
yielding methylated aglycone and methylated sugars. The
methylated aglycone was identified as 7,4'-dihydroxy, 5,8-
dimethoxy flavanone and the methylated sugars were identi-
fied as 2,3,4-tri-O-methyl L-rhamnose 2,4,6-tri-O-methyl D-
16. B.C. Saunder and F.G. Mann, Practical Organic Chemistry, New York,
Longman, p. 365 (1936).
17. Y.R. Deng,A.X. Song and H.Q.Wang, J. Chin. Chem. Soc., 51, 629 (2004).
18. N. Song, W. Xu, H. Guan, X. Liu, Y. Wang and X. Nie, Asian J. Trad.
Med., 2, 218 (2007).