PHYTOCHEMICAL INVESTIGATION OF ABUTILON INDICUM

By K. N. GAINDand K. S. CHOPRA

Abstract

A b u t i l o n i n d i c u m ,'dried and powdered aerial parts, was extracted with

petroleum ether. The petroleum ether extract, Fraction A, was sapofiified. T h e
unsaponifiable matter was found t o contain n-alkane mixture (C22-C34)Jan
alkanol fraction and /3-sitosterol. The marc was extracted with alcohol 50°/o and
the extract fractionated into Fractions B, C and D. T h e residual plant material
was extracted with water and the extract fractionated into Fractions E and F. In
Fraction B, vanillic, p-coumaric, p-hydroxybenzoic, caffeic and fumaric acids, and

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in Fraction C, p-P-D-glucosyloxybenzoic acid and gluco-vanilloyl glucose were
isolated and identified. In fractions D and F, fructose, galactose, glucose, leucine,
histidine, threonine, serine, 'glutamic acid and aspartic acid were identified by
CO-chromatography.In Fraction E,' a mucilage, the acid hydrolysate showed
galactose and galacturonic acid.

Abutilon indicum (Malvaceae) grows in India, Malaya, Philippine Islands, and

Indo-China where it is claimed to be of therapeutic use as febrifuge, anthelmintic,
anti-emetic, anti-inflammatory and in urinary and uterine discharges, piles and
lumbago. Seeds of A. indicum have been reported to contain semidrying oil
composed of linoleic, linolenic, oleic, palmitic and stearic acids. T h e unsaponi-
fiable matter contained 8-sitosterol. Raffinose was found t o be present in alcoholic
extract of defatted seeds [ l , 21. Cyanidin-3-rutinoside has been identified and
gossypetin-8-glucoside isolated and identified in petals [3].

Table 1
Response to chemical tests of extractives of A. indicum
- -

Extractive Sterols Carbo- Glyco- Acidic Phenolic Amino

(010 w/w) hydrates sides compounds compounds acids

Petroleum ether (2.49) + - - - - -

Benzene (0.24) + - - - - -
Chloroform (0.97) - + + + + -
Ether (0.64) - + + + + -
Acetone (2.72) - + + - + -
Alcohol (4.18) - + + - + +
Water (15.29) - + - - - +
 Investigation of Abutilon indicurn 175

ABUTILON INDICUM
3.375 kg
extracted with petroleum ether

I I
Marc (1) Petroleum ether extractive
I
extracted with alcohol 500/0 (vlv)
FRACTION A, 2.2OIo

I
I
Marc (II)
I
Alcohol extractive

extracted with water

I
suspended in water, acidified with dil.

'
satura.ted with chloroform sulphuric acid and extracted with ether

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Aqueous extract Marc (III)
I
Aqueous Acidic portion
Ether
rejected
I extractive

+,
treated with neutralized with CaCO.,. FRACTION B, 0.32~10
".
Acetone (2 vols) - concentrated and
extracted with n-butanol

. Filtrate
1I
Evaporated to dryness
Precipitate
FRACTION E, l.lO/o 1I
Aqueous portion Butanol
extractive
treated with alcohol FRACTION C, 0.9"/0
I
FRACTION F, 0.65°/o 95010 (vlv) to make final
concentration of alcohol

I I
Filtrate Precipitate
1
Eva'porated to dryn'ess
rejected

I
1 FRACTION D, l.OOlo
Scheme 1. Fractionation of constituents of the plant rnaterial A. INDICUM

The dried and coarsely powdered plant rnaterial consisting of stem, leaves, and flowering tops
was extracted by Soxhlet extraction with organic solvents; extraction with water was done by
maceration. The percentage extractives and tests for the presence of chemical constituents of
176 Gaind and Chopra Planta rnedica Vol. 30 1976

usual occurrreiice are shown in Table 1; fractionation of various constituents was carried as givcn
in Scherne 1.

Results and Discussion

Fraction A
T h e unsaponifiable inatter of fraction A when eluted on an alumina column
gave the following products.
Product 1, 2.3O/o, m.p. 4S0; i.r. spectrum bands a t 2945,2845 cm-' (CH, stretch-
ing) 1460 cm-1 (CH, bending) and a doublet a t 720, 710 cm-' (CH, rocking); no
absorption in the O H region. The i.r. spectrum was suggestive of the product to
be a straight chain alkane of seven o r more carbon atoms [4]. GLC analysis
showed that the product is a mixture of a t least 13 components, C,,-C,,, which

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were identified by comparing with gas chromatogram of a mixture of authentic
sample of n-alkanes (Table II).

Table II
Retention time and per cent composition of constituent n-alkanes of Fraction A

Carbon Retention time (min) Composition (per cent)

traces
1.0
3.0
1.9
12.8
40.3
9.3
16.4
3.2
10.2
traces
traces
traces

Product I I , O.SO/o, m.p. 85.5O, i.r. spectrum of the product showed absorption
bands a t 3300 cm-' (O-H bonded stretching), 2910 cm-' (CH, stretching), 2845
cm-' (CH, stretching), 1460 cm-' (CH, bending), 1055 cm-' (C-O stretching) and
725, 715 cm-' (CH, rocking). T h e i.r. spectrum was suggestive of a long chain
primary alcohol [7]. T h e acetate derivative showed absorption band in the i.r.
spectrum a t 1740 cm-' (C-O stretching of acetate). There was n o absorption in
the O H region.
 Investigation of Abutilon indicum 177

Product 111, 1.4010,m.p. 132-140°, gave positive tests for sterols, mixed melting
point with an authentic sample of B-sitosterol remained undepressed. Acetate,
m.p. 127-129O. The i.r. spectra of the product and its-acetate were superimposable
with those of B-sitosterol and B-sitosterol acetate.

Fraction B -
Fraction B was investigaged by PC; the spots gave characteristic colors with'
diazotized p-nitroaniline, sulfanilic acid reagents and also responded positively
to bromophenol blue (Table III). The CO-chromatographicinvestigation showed
four spots resembling in Rr values and colors to those of vanillic, p-coumaric,
p-hydroxybenzoic and caffeic acids; Products IV to VII. These acids were isolated
by preparative paper diromatography; Whatman 3 mm paper; benzene-acetic
acid-water (10:5:3). The U.V. and i.r. spectra of the isolates were comparable with
those of authentic specimens.

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T a b l e III
RI X 100 values and color* of spots shown by chromatograms of Fraction B

@ Solvent systems*" Detection Reagentse** [S, 61

Spot 1 II III 1 2 3
No.
1 86 26 84 V tP tV
2 64 38 86 B R tP
3 51 30 88 Ro Y -
4 43 61 - V Or -
5 26 88 25 fR Y -
6 20 76 - fR O rY -
7 1O 14 80 Br Or tG
f: faint, t: transient, B: blue, V: violet, Ro: rose, R: red, Br: brown, P: pink, Y: yellow,
Or: orange, G: green.
*'$ 1 Benzene-acetic acid-water (10:5:3)
II Isopropanol-ammonia (0.88)-water (20:1:2)
III Butanol-pyridine-water (14:3:3)
:'$.:-" 1. Diazotized p-nitroaniline reagent
2. Diazotized sulfanilic acid reagent
3. Ferric chloride reagent.
@ Rf and colors of spots 1, 2, 3 and 7 were comparable with those of authentic specimens of
vanillic, p-coumaric, p-hydroxybenzoic and caffeic acids respectively.

Sublimation of fraction B yielded another Product VIII, which perhaps escaped

being revealed on the chromatogram due to volatilization during drying which is
done before subjecting the paper to the spray reagents.
178 Gaind and Chopra Planta medica Vol. 30 1976

Product VI11, m.p. 282O (sealed capillary), gave positive tests with solutions of
bromophenol blue and sodium bicarbonate. The properties and melting point of
the product was comparable with that of furnaric acid. The i.r. spectra of the two
were superimposable. The melting points of admixture of the product and fumaric
acid showed no depression and so also that of the product of methylation of the
former ( m q . 100°) and dimethyl fumarate.

Fraction C
Fraction C was chromatographed on a column of silica gel eluting with ether
and methanol. The ether eluate was designated product IX and the other as
Product X.

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Product lx, 4.1010, m.p. 209-210°, [a]2,7-81° (c 2.41). It was liydrolyzed in
acid medium. One of the products of hydrolysis, IX A, m.p. 212-213O, on PC
and TLC analysis was found to be comparable with p-hydroxybenzoic acid. It
showed similarity of m.p., i.r. and U.V. spectra with that of authentic sample of
the acid. The rnass spectrum showed parent ion peak at mle 138 and other
fragments a t mle 121, 93, 65, 39 were found to conform t o published spectrum
of p-hydroxy benzoic acid [a].
The second product of hydrolysis, IX B, gave positive tests for carbohydrrites.
Chrornatographic investigation revealed a single entity; Rr value comparable with
that of authentic D-glucose; phenylosazone derivative, m.p. 20g0, mixed m.p.
with authentic osazone of D-glucose undepressed.
The product IX was thus considered to be a glucoside of p-hydroxybenzoic
acid. The rnelting point (209-210°) was closely comparable with that of p-P-D-
glucosyloxybenzoic acid (lx)(reported m.p. 211-212O) [9].
The position of linkage of sugar moiety to the aglycone and elaboration of
structure was further established by making a propyl derivative and subsequent
acetylation. The m.p. 122-123O of the resulting product, IX C, was comparable
with that of propyl ester of p-j3-D-tetraacetylglucosyloxybenzoicacid (reported
m.p. 124.5-125.j0) [IO]. The elemental analysis, i.r. and nmr (Fig. 1) spectra
supported the formation of the above derivative. In the mass spectrum analysis
of IX C , the parent ion peak was found to be missing which is, however, not
unusual for such type of cornpounds [ I l ] . The fragmentation pattern (Scheme 2)
describes the peak at mle 450 due to 'the primary ion obtained by cleavage at the
ester bond and loss of C,H,O followed by splitting off of glycosidic linkage. The
 Investigation of Abutilon indicum
179

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180 Gaind and Chopra Planta medica Vol. 30 1976

resultant tetraacetyl glucose oxonium ion is shown by a prominent peak a t mle

331. The fragmentation pattern, of tetraacetylglucose oxoniurn ion into daughter
ions is discussed in a subsequent para. The data of analysis of products IX, IX A,
IX B and IX C support the chemical structure of IX, contemplated a t p-p-D-
glucosyloxybenzoic acid.

OAC

CH OAC

+
- 0 = - CHOCO

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OAC
-ml: 43
-
m/g 169 !$/-(09
- 331
m/o

H o C o + (12 1 -Co) (9 3 - CO) (65-C2Hz)

93 ?le 6 5 m k 39
?/O 121

Scheme 2. Scheme of fragmentation of product IX C

Product X , 27.O01o, m.p. 226-228O (decomp.)

The product X on hydrolysis in acid medium yielded aglycone, X A, and a
sugar moiety, X B. The aglyi'one gave positive tests characteristic of phenolic O H
and C O O H groups. Melting point (208O), chemical tests, ~hromatographic
analysis, i.r. and U.V.spectra showed the product X A comparable with vanillic
acid. The mass spectrum showed parent ion peak at rnle 168. Other peaks were
found as expected for the breakdown of phenol aromatic acids and ethers [8].
The sugar moiety X B was identified as D-glucose in the same way as in the case
of product IX B.
The product X was considered to be glucoside of vanillic acid. The acetyl
derivative, product X C, was subjected to analysis. Product X C, m.p. 157-158°,
[a12 -42.88 (CHCI,, c 1.66); calcd. for C3BH44022. 112C3H70H, M.W. 858.8.
C, 52.44; H, 5.63; CH,CO, 40.0; CH,O, 3.61°/o; Found: C, 52.93; H. 5.21;
CH,CO, 39.0; CH,O, 3.22O/o, M.W. 853, nmr (Fig. 2). The elemental analysis,
and mass spectrum (Scheme 3) of X C showed the presence of two molecules of
glucose for each molecule of the aglycone, vanillic acid. In the mass spectrum the
fragment of mle 331 was suggestive of tetra-acetylglucose oxonium ion. The mle
 Investigation of Abutilon indicum
186

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182 Gaind and Chopra Planta medica Vol. 30 1976

498 fragment indicated loss of tetra-acetylglucose moiety either at the para

position or at the ester linkage of the sugar with the carboxyl group of the acid.
Since the hydrolytic studies of product X and mass spectral analysis of product
X C failed to reveal the presence of disaccharide moiety, the attachment of one
molecule of tetraacetyl glucose at the para position and another with carboxyl of
vanillic acid is likely to be as shown in Scheme 3.

C H,O

C-O

A c 0
OA c OAc

X C(M=B28) .

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CH p

OAc OAc
- 498 -m/0498

Scherne 3. Scheme of fragmentation of product X C

The data of analysis of products X A, X B, and X C, support the structure of

product X as gluco-vanilloyl glucose. Glucovanilloyl glucose, Picrorhizin, is
reported earlier to be present in rhizomes of Picrorhiza ktlrroo [12].
In the foregoing discussion of mass spectra of products IX C and X C, some
characteristic features were observed which had been reported previously by
PEARLand DARLING[ I l ] who made an exhaustive study of application of mass
spectrometry in the structure elucidation of acetates of salicin (XI), salirepin (XII)
and ~alicyloylsalicin(XIII).
It was shown that primary fragmentation occurred at C-1 carbon of the glucose
moiety resulting in a prominent peak at mle 331 of tetraacetyl glucose oxonium
ion. The subsequent fragrnentati,on took place through elimination of acetic acid
(-CH,COOH), ketene (-CH,=C=O) and again acetic acid.and showed resultant
ion peak at mle 169. The intermediate fragment, as described'by B U D ~ I K I E W I C Z
 Investigation of Abutilon indicum 183

-p
Q
~
:o O-c\
XII1 I
~-i O XIV
=H3

229
OAc

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et al. [13] produced as a result of loss of acetic acid and ketene is also shown
by the peak at mle 229 (XIV) in the mass spectral analysis of products IX C and
X C . T h e decomposition of tetraacetylglucose oxonium ion also takes place by
elimination of ~ W Omolecules of acetic acid followed by ketene resulting in the
same fragment of mle 169. The intermediate fragment (XV) at mle 211 produced
by loss of two molecules of acetic acid is also shown in the data of mass spectral
analysis of both the products. Further breakdown of fragment mle 169 takes place
by elimination of one molecule of acetic acid, resulting in mle 109..
It has also been reported by PEARLand DARLING[ I l ] that a weak molecular
ion peak was characteristic of al1 the glucoside acetate spectra studies. The
absence of molecular ion peak in spectra of products IX C and a very weak peak
(M-1) in that of product X C is, therefore, not exceptional.

Fraction D
A 2 per cent aqueous solution of fraction D, was chromatographed on Amberlite
IR-120 (H) and Amberlite I R 4 5 (OH) in succession. The effluent was dried
under vacuum and reserved for investigation as Product XVI. The columns of
Amberlite IR-120 (H) and I R 4 5 (OH) were eluted with 5 per cent solution of
ammonia and 1.0 per cent hydrochloric acid respectively. The effluents were dried
under vacuum and the residues mixed and labelled as Product XVII.
Product (XVI), gave positive Molisch test. TLC investigation on silica gel G
showed three spots; isopropanol-ethyl acetate-water 7:1:2 (RI 0.81, 0.61, 0.60)
and ethyl acetate-acetic acid-methanol-water, 12:3:3:2 (Rr 0.64, 0.60, 0.36).
Fructose, galactose and glucose showed comparable Rt values in al1 the syst.ems.
Product (XVIZ),gave positive tests for amino acids. PC investigation showed
seven spots. Phenol (saturated with water), (Rr 0.80, 0.66, 0.50, 0.38, 0.29, 0.20,
184 Gaind and Chopra Planta medica Vol. 30 1976

0.14); n-butanol-acetic acid-water, 25:6:25 (RI 0.69, 0.38, 0.36, 0.34, 0.19);
pyridine-iso-amyl alcohol-water-diethylamine, 100:100:70:3 (Rr 0.84, 0.54, 0.46,
0.42, 0.34, 0.28, 0.18). Authentic samples of leucine, histidine, threonine, serine,
glutamic acid and aspartic acid showed comparable Rt values and colors with six
of the spots shown by products (XVII) on CO-chromatography.

Fraction E
Fraction E gave positive Molisch test; soluble in water forming viscous solution;
insoluble in alcohol, acetone; gave negative Ninhydrin and Millon's tests. An acid
(2N HCl) hydrolysate of fraction E on PC showed eight spots; iso-propanol-
water; 4:l (Rt 0.81, 0.71, 0.63, 0.59, 0.54, 0.12, 0.09, 0.04); and on TLC five
spots; iso-propanol: ethyl acetate: water, 7:1:1, (Rr 0.93, 0.85, 0.81, 0.61, 0.60).
Authentic samples of galactose and galacturonic acid showed comparable Rt
values and colors on CO-chromatographyin PC (Ri 0.54 and 0.12 respectively)

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and in TLC (RI 0.60 and 0.85) with two of the above spots.

Fraction F
Fraction F gave positive Molisch and Ninhydrin tests, ash 72010. The fraction
F was extracted with alcohol 70°/o. The chromatographic investigations showed
similar results as in the case of fraction D.

Acknowledgement

We are grateful to Professor J. W. STEELE,Acting Dean, Faculty of Pharmacy, The University

of Manitoba, Winnipeg, Canada and Dr. K. L. MUNSHI,Central Drug Research Institute,
Lucknow, India, through whose courtesy GLC, NMR and Mass spectra were obtained.

References

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 investigation of Abutilon indicum 185

[9] SABALITSCHKA, T. and SCHWEITZER, F. L.: Arch. Pharm., 267, 675 (1929); through Chem.
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Address: Dr. K . N . Gaind and Dr. K . S. Chopra, Department of Pharrnaceutical Sciences,

Panjab University, Chandigarh 160014, INDlA

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