Isolation of secondary products from Ipomoea digitata a medicinally important plant

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D. Madhavi1, B. Rama Rao2,
Peddolla Sreenivas3, G.L. David Krupadanam3,
P.Maheswari Rao1,   K. Janardhan
Reddy4 and P.B. Kavi Kishor1*

*For Correspondencepbkavi@yahoo.com

1Department
of Genetics, Osmania University, Hyderabad
- 500 007,India

  2Natural
Products Lab, Indian Institute of Chemical Technology, Hyderabad
500 007,India

3Department
of Chemistry, Osmania University, Hyderabad-
500 007,India

4Department
of Botany, Osmania University, Hyderabad
- 500 007, India

Abstract

The paper reports the isolation and characterization of β-sitosterol,
t-cinnamic acid [undecyl(E)-3-(4- hydroxyphenyl)-2-propenoate], an unknown coumarin and a lignan type resin glycoside
from the tuberous roots of Ipomoea digitata. The structures of the compounds were
elucidated on the basis of extensive chemical and spectroscopic data.
Importantly, one of the compounds exhibited significant antibacterial activity
against Pseudomonas aeruginosa and E coli. The resin glycosides are known
as purgative ingredients and hence have medicinal value. However, the exact chemical
and biological activity of the resin glycoside isolated in this study is yet to
be ascertained.

Key words: Ipomoea digitata, Convolvulaceae,
Undcyl (E)-3-(4-hydroxyhenyl)-2-propenoate, Coumarin, Resin glycoside

Introduction

Ipomoea digitata is a wide-spread, paleotropic,
perennial herb belonging to the family Convolvulaceae.
It is commonly known as wild yam or Vidari Kanda in Telugu. The tuberous roots are used as tonic,
aphrodisiac, cholagogue, demulcent, diuretic, emmenagogue, galactogogue and rejuvenant. Aqueous infusions of the roots are used in
Indian traditional medicine for treating epileptic seizures and as antioxidative in Ayurvedic
medicine. Hundred grams of Dabur
Chavanprash contains 1.195 g ofI. digitata root powder. Studies on some
other species of this genus revealed the presence of Ipangulins,
the first pyrollizidine alkaloids, ergoline alkaloids, stoleniferins
and resin glycosides (1-6). Recent reports revealed the presence of resin
glycosides in the leaves and stems ofI. digitata (7). The aerial parts are also used as folk
medicine inJapan (8). Chemical constituents of the root of this plant (scoperon,
β-sitosterol, taraxerol)were reported earlier by Rao et al. (9). The
pharmacological activity of the fraction D (10) of this plant extract has drawn
our attention to the chemical and biological study of this plant. One of our
efforts to discover the structurally diverse and biologically significant
metabolites fromI. digitata has
led to the isolation of two known (named as compounds
1, 2) and two unknown compounds (compounds 3 and 4) with spectral and chemical
data. We also report, antibiotic activity of compound 3 on gram-negative
bacteria Pseudomonas and E. coli.  

Materials and Methods

Fresh
tuberous roots of Ipomoea digitata were collected from Nallamalais
forests ofAndhra Pradesh, India. The plant specimen was
deposited in the herbarium of the Department of Botany, Osmania
University,Hyderabad, India. Three kilograms of fresh
tuberous roots were cut into small pieces and dried under shade. The dried tuberous
roots were pulverized by using a mixer and extracted in hexane by dipping it
for 3 days, followed by re-extraction in methanol. The hexane extract was
filtered and concentrated under reduced pressure. The methanol extract gave 83 g
of residue after concentration under reduced pressure. With the aim of
isolating polar compounds, the dried root powder (1 kg) was taken freshly and
extracted in water for 3 days. Hexane extract was subjected to silica gel
column chromatography, eluted with a solvent system of hexane/ethyl acetate to
separate the compounds from the mixture. The fraction (10 g) eluted by hexane
and ethyl acetate (4:96) resulted in the isolation of a pure compound (compound
1) as determined by thin layer chromatography (TLC). This compound was further
analyzed by nuclear magnetic resonance (1H NMR) and mass
spectrometry (MS) for identification. The fraction eluted by hexane
: ethyl acetate (95:5) was repeatedly
subjected to silica gel purification which afforded 65 mg of the compound. The
white powder was successively subjected to 1H NMR, MS, infra red (IR) and 13C NMR analysis for structure
determination (compound 2). The fraction (28 g) of methanolic
extract, eluted with chloroform : ethyl acetate (80:20), gave compound 3. Upon
extracting this in hexane and benzene (50:50) for 12 h, yellow coloured needles were obtained. The compound was then
subjected to 1H NMR, MS, IR and 13C NMR. The dried water
extract was concentrated and extracted again in chloroform (2 x 500 ml) to
remove low polar compounds. The remaining residue dissolved in methanol
consisted of a single compound that was confirmed by TLC. The methanolic extract (1.5 g) was dried and concentrated to
give a semi-solid sticky material, which was separated by column chromatography
on silica gel (100:200 mesh). This was separated with chloroform
: methanol (50:50) solvent system in the column and designated as
compound 4.

The IR spectra were recorded on a Bruker Tensor 27 FT- IR
spectrometer with KBr pellets. The 1H NMR
spectra were recorded at 400 MHz (Varian make). 
The 13C NMR spectra were performed in CDCl3 at
75.5 MHz for compounds 1 and 2 but 13C NMR of compound 3 was noticed
in dimethyl sulphoxide (DMSO) at 75.5 MHz. Electron ionization-MS were obtained at 70 ev
and fast atom bombardment (FAB)–MS using Argon (6 kv)
as the FAB  gas. Silica gel (60-120 mesh
and 100-200 mesh, Merck,Darmstadt, Germany) was
employed for column chromatography. TLC was carried out on pre-coated kiesel gel 60 F254 (0.25 thick, Merck,Darrnstadt, Germany) plates, with hexane-ethylacetate and chloroform–ethylacetae
as solvent systems. Coloured spots were visualized by
exposure to iodine vapours followed by spraying with
10% sulfuric acid solution. Dragendorff”s reagent was
used for the detection of alkaloids. High performance liquid chromatography (HPLC,
Eclipse XDB-C-18 column, 5 µm, 4.6 x150 mm, 25%-100% methanol in water over 8
min followed by 100% methanol to 11 min, 1 ml/min, 30 0C) was
carried out in combination with TLC. Methanol, chloroform, ethyl acetate and
hexane which were used for extraction of compounds were purchased from the Finar chemicals,Hyderabad.
Methanol-sulfuric acid, Dragendorff reagent,
ethanol-sulfuric acid were used as reagents to detect the compounds that are
separated on the chromatogram. Silica coated glass plates for TLC were
purchased from Merck. All other reagents and chemicals used were of analytical grade.

Antibacterial
activity was tested against gram negative bacteria like Pseudomonas and E.coli by
single disc method (11). Test
bacterial strains were cultured in sterilized Luria Bertani (LB) broth (pH 5.7)
for 16-18 h at 37 0C on a rotary shaker. LB media with agar as
gelling agent was prepared and sterilized at 121 0C for 15-20
minutes in an autoclave. Plating was carried out under aseptic conditions. A
loop full of liquid broth of bacterial culture was spread over solidified LB
media. Sterilized discs (Whattmann filter paper) were
placed at equal distances. The four compounds each of 5 mg were weighed and
dissolved in 5 ml of DMSO. Dissolved compounds (3 µl) were placed on the discs
and plates were wrapped tightly with parafilm and
incubated at 37 0C for 24 h.

Results and Discussion

Extraction of tuberous root powder of Ipomoea digitata
resulted in the isolation of four compounds. The first compound is a smooth, whitish pink, amorphous
powder. The structure (Fig. 1) of this compound (β-sitosterol)
was determined based on the spectral data like 1H NMR and Mass (Fig.
2) and also correlated with the previously available data (12). Compound 2,
isolated for the first time from I. digitata, is a white powder, with a melting point of 80
oC, chemical formula of C20H30O3
and is identified as undecyl-(E)-3-(4-hydroxyphenyl)-2-propenoate
(trans-cinnamic acid). Its structure and 13C
NMR spectrum are shown in figures 3 and 4 respectively. The third compound
appeared as a pale yellow coloured needles with a
melting point of 205 oC and identified as 5-hydroxy-7-methoxy
coumarin based on its spectroscopic data. Its
structure and 1H NMR spectrum are shown in figures 5 and 6. It
exhibited the activity under ultraviolet light. Compound 4 is soluble only in
methanol and water, and is a brown coloured, sticky,
semi solid, resin type glycoside. The signals in 1H NMR (Fig. 7) and
Mass spectra allowed us to conclude that this compound is a resin glycoside.
The 1H NMR and Mass spectral signals of compound 4 did not show any
correlation with the available spectral data (7), indicating that this compound
is different from the compounds already isolated from the leaves and stems of I. digitata
earlier. However, the exact physical nature and chemical structure of the glycoside
is yet to be determined.

Structure of β-sitosterol

Fig. 1. Structure of β-sitosterol

Mass spectrum of â-sitosterol,Structure of t-cinnamic acid

Fig. 3. Structure of t-cinnamic acid (undecyl (E)-3-(4- hydroxyphenyl)-2-propenoate)

CNMR spectrum of t-cinnamic acid, Structure of 5-hydroxy-7-methoxy coumarin

Fig. 5. Structure of 5-hydroxy-7-methoxy coumarin

H NMR of 5-hydroxy-7-methoxy coumarin

Fig. 6. 1H NMR of 5-hydroxy-7-methoxy coumarin

H NMR spectrum of lignan type resin glycoside

Fig. 7. 1H NMR spectrum of lignan type resin glycoside

None of the above compounds showed any antioxidant activity though extracts of this
plant are used in Ayurveda as an antioxidant. However,
coumarin, when tested against Pseudomonas aeuriginosa and E. coli, inhibited the growth of these
bacteria but not others. This is the first time that bacterial activity has
been found for the coumarin isolated from this plant.
Hence,I.digitata can
serve as a good source for the isolation of this bioactive compound. The
inhibition zone (14 mm) of bacterial growth was observed around the disc
containing the compound. Many workers reported major effects like antinociceptive, antifungal and anticancer activities for
the compounds of Ipomoea genus (13-15).
The resin glycosides, originated from the Convolvulaceous
plants, are well known as the purgative ingredients in some traditional
medicines. They are divided into ether-soluble resin glycosides “jalapin” and ether-insoluble resin glycoside “convolvulin” (16). Resin glycosides have also recently been
isolated and characterized from the leaves and stems ofI. digitata (7). They named it as digitatajalapin I since it was isolated from jalapin fraction. The resin glycoside isolated in the
present study is ether-soluble type. Therefore, it could be a “jalapin” related glycoside. However, the biological effects
of the resin glycoside isolated in this study needs to be determined, which is
underway.

Acknowledgement

Ms.D. Madhavi would like to thank CSIR,New Delhi for financial
assistance in the form of fellowship.

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