Basu et al.

Chemistry Central Journal (2017) 11:118

DOI 10.1186/s13065-017-0349-2

RESEARCH ARTICLE Open Access

Polysaccharides from Dolichos biflorus

Linn and Trachyspermum ammi Linn seeds:
isolation, characterization and remarkable
antimicrobial activity
Shibani Basu1, Manojit Ghosh2, Rupam Kumar Bhunia3, Jhuma Ganguly1* and Bimal K. Banik4*

Abstract
Polysaccharides are structurally complex and essential constituents of life, and therefore, studies directed to these
kinds of molecules have received scientific attention. Despite an easy availability of Dolichos biflorus Linn and Tra-
chyspermum ammi (Linn) seeds isolation, characterization and antimicrobial studies of polysaccharides derived from
these two natural sources have not been investigated. Therefore, we report here isolation of polysaccharides, their
purification and characterization from Dolichos biflorus Linn and Trachyspermum ammi (Linn) seeds. Gel permea-
tion chromatography, GC–MS, SEM, XRD, EDX and FT-IR analyses show the presence of three pentose sugar such as
d-ribose, d-arabinose, d-xylose and hexose sugar such as d-mannose, d-galactose and d-glucose. Unprecedented anti-
microbial activity of these polysaccharides against Gram positive bacteria such as Staphylococcus aureus and Bacillus
subtilis and Gram negative bacteria such as Escherichia coli and Pseudomonas aeruginosa are established.
Keywords: Polysaccharides, Structural elucidation, Chemical characterization, Physiochemical identification,
Antimicrobial activity

Introduction nature of Trachyspermum ammi (Linn.) Sprague and

There has been tremendous interest in the use of medici- Dolichos biflorus Linn in detail. Literature is found less
nal plants in developed as well as developing countries, secured on the elucidation of their structures of purified
because compounds obtained from medicinal plants have extracts of these species and studies of their significant
been shown to be effective sources of therapeutic agents, biological activity.
without undesirable side effects [1, 2]. Polysaccharides Trachyspermum ammi (Linn) Sprague (Ajowan),
are very crucial since they have tremendous medicinal belongs to the family of Apiaceae. Also known as bish-
values. Current studies have proved that the structures op’s weed, it is an aromatic spice closely resembling
of polysaccharides are closely related to their biological thyme in flavor [4]. It is a native of Egypt and is distrib-
activities [3]. So elucidation of their structures is a fun- uted in the Mediterranean region and South-west Asia.
damental objective for understanding structure–activity It has long being used as the principal source of thymol T.
relationships and cause of these biological activities. ammi seeds and is employed as an antiseptic, aromatic,
We report here an analysis of the structural composi- carminative and antioxidant source [5]. Its oil is used in
tion of purified polysaccharides extracts and chemical the preparation of lotions and ointments in cosmetics
industries and as a spice in many food preparations [6]. It
*Correspondence: jhumaiiest@gmail.com; bimalbanik10@gmail.com
has been reported to possess strong insecticidal activity,
1
Department of Chemistry, Indian Institute of Engineering Science bronchodilatory effect on asthmatic airways and analge-
and Technology, Shibpur, Howrah, West Bengal, India
4
sic effect [7–9].
Research & Education Development, Community Health Systems
of South Texas, 3135 South Sugar Road, Edinburg, TX 78539, USA
Dolichos biflorus Linn (Horsegram or Kulthi) is well
Full list of author information is available at the end of the article known throughout India as a draught resistant crop. It

© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Basu et al. Chemistry Central Journal (2017) 11:118 Page 2 of 10

also enriches the soil with nitrogen. So after harvesting samples were dissolved in deionized water and applied to
the seeds, the soil can be ploughed with green manure a Sephadex G-25 column (600 × 16 mm) that had previ-
[10]. Dolichos biflorus is a well known medicinal plant ously been equilibrated with Mili-Q water at a flow rate
for its folk- medicinal properties. In herbal medicine, the of 0.3 mL min−1 and the column was then eluted with of
seeds of it are mainly used as tonic, astringent, diuretic, Mili-Q water. Collected fractions were identified by phe-
and are also recommended in asthma, bronchitis, urinary nol–sulphuric acid assay at 490 nm for the estimation of
discharges, hiccoughs, heart trouble and other diseases of neutral sugar [14].
the brain [11–13].
After isolation, both samples from the seeds of T. ammi Compositional analysis
and D. biflorus were purified, analyzed and compared by The polysaccharides were hydrolyzed by TFA and derived
chemical and physico-chemical route. The percentage of by “acetic anhydride-pyridine” method. The derivatives
active ingredients in the purified extract from the natu- were used to determine the monosaccharide composi-
ral samples is dependent on geographical distribution as tions, because monosaccharides, in these conditions,
well as the environmental conditions such as tempera- were converted to volatile substances and therefore,
ture, rainfall, altitude, and hr of sun light exposure. they were easy to be detected by gas chromatography–
mass spectrometry. Analytical GC and GC–MS were
Materials and methods performed for quantitative and qualitative analysis of
Sample preparation monosaccharides present in the purified fraction using
Seeds of Dolichos biflorus Linn and Trachyspermum alditol acetate derivatives with inositol as an internal
ammi (Linn) Sprague were obtained from West Bengal, standard [15]. Samples (200 µg) were hydrolyzed with
India and identified (Fig. 1, Table 1). Seed samples were 2N TFA (trifluoroacetic acid) at 120 °C for 2 h, ­NaBH4
prepared by extracting 10 gm of seeds of each sample (sodium borohydride) was used for reduction followed
separately in 50 mL of water and stirring for 48 h at room by 1 h incubation at 100 °C with acetic anhydride and
temperature (25 °C). The samples and named as TAE (T. pyridine (1:1). The hydrolyzed monosaccharides were
ammi water extract) and DAE (D. biflorus water extract). then extracted with DCM (dichloromethane), analyzed
on Agilent 6820 gas chromatograph equipped with HP-5
Isolation and purification of water soluble carbohydrate fused silica capillary column (30 m × 0.25 mm I.D). Mass
The crude aqueous extracts were purified by gel per- spectra quantified on GC 7890A series equipped with
meation chromatography (GPC). The freeze-dried MS 7000 GC/MS triple quad using DB-5 silica capillary

Fig. 1 Seed of Trachyspermum ammi (Linn) Sprague (a) and seed and Dolichos biflorus Linn (b)

Table 1 Identification and authentication of samples

Sl. no. Specimen/sample no. Scientific name Common name Family name

1 J.G.1 Trachyspermum ammi (Linn) Sprague Ajwain, ajoan, carom Apiaceae; Alt. Umbelliferae
2 J.G.2 Dolichos biflorus Linn Horse-gram, kulthi, hurali, Madras gram Fabaceae; Alt. Leguminosae
Basu et al. Chemistry Central Journal (2017) 11:118 Page 3 of 10

column (30 m × 0.25 mm I.D). Nitrogen was used as the TGA−DTA analysis
carrier gas (1.5 mL min−1). Detection was done by FID Thermal analysis gives properties like enthalpy, thermal
(300 °C) and identification by mass spectrometry held at capacity, mass changes and the coefficient of heat expan-
250 °C. sion. TGA and differential scanning calorimetric (DTA)
analysis were carried out by Perkin Elmer SII, Diamond
FT‑IR analysis TG/DTA machine. The samples were heated to 600 °C at
The major structural groups of purified extracts were a heating rate of 10 °C min−1, under flowing nitrogen of
detected using Fourier transform infrared spectroscopy 10 mL min−1.
(FT-IR). The FT-IR spectra were recorded in the region of
4000–400 cm−1 on a JASCO, FT/IR-460 PLUS using KBr Study of antimicrobial activity
pellet method. Background correction was made using a The disc diffusion method was applied to evaluate the
reference blank KBr pellet. The purified polysaccharides antimicrobial activity with four human pathogenic bac-
were ground with KBr powder by mortar and pestle. The teria. Gram positive bacteria such as Staphylococcus
mixture was then pressed into pellets for FTIR measure- aureus, Bacillus subtilis, Gram negative bacteria such
ment in the frequency range of 4000–500 cm−1. as Escherichia coli, Pseudomonas aeruginosa were used
in this study. 100 mL conical flask of nutrient broth was
SEM and EDX analysis inoculated with the test organisms and incubated at 37 °C
The samples were used for Scanning electron micros- for overnight. By using a sterile pipette, 0.6 mL of the
copy (SEM) analysis by fabricating a drop of suspension broth culture of each test organism was added to 60 mL
onto a clean electric stubs and allowing the solvent (i.e. of molten agar, which was cooled at 45 °C, mixed well
water, methanol) to completely evaporate with platinum and poured into a sterile Petri plate. Normal saline water
coating. The morphology of the purified extracts was was used as negative control and Tetracycline was used
observed on a ZEISS EVO 18 electron microscope with as positive control in comparison with test organisms.
an accelerated voltage of 10–20 kV. Then, the plates were left at room temperature for 2 h
Elemental analysis of purified extracts was carried out to allow diffusion of the test sample and incubated face
using energy dispersive X-ray spectroscopy (EDX). The upwards at 37 °C for overnight.
EDX of the purified extracts were measured by ZEISS
EVO 18 electron microscope. Results and discussion
Isolation and purification by gel permeation chromatog-
XRD analysis raphy (GPC) on a Sephadex G-25 column (600 × 16 mm)
X-ray diffraction (XRD) analysis of drop-coated films of obtained UV actives region (at 490 nm) peaks for the
extracts was prepared for the study of phase and nature. phenol sulfuric acid assay (Fig. 2) from TAE and DAE
A Bruker D-8 Advanced X-ray diffractometer with scan- (extracts were eluted with deionized water). As shown
ning range from 2θ = 20° to 80° and Ni-filtered Cu Kα in Fig. 2, absorbance at 490 nm was observed, confirm-
radiation with wavelength 1.540598 Å used for this ing that the isolated fractions were mainly polysaccharide
characterization. rich.

Fig. 2 UV spectrophotometry analysis (the UV actives region at 490 nm) for the purity of water soluble polysaccharides of T. ammi (Linn) Sprague
(a) and D. biflorus Linn. (b) purified by gel permeation chromatography
Basu et al. Chemistry Central Journal (2017) 11:118 Page 4 of 10

GC–MS analysis determined six kinds of monosaccha- ratios of the monosaccharide constituents in TAE were
rides in TAE and four kinds of monosaccharide in DAE found to be present at 6:7:3:38.2:35.1:10.5 (Table 2).
by comparing with standard sugar peak (Fig. 3). The GC On the other hand, by the GC–MS analysis in DAE
and GC–MS analyses showed the presence of three pen- (Fig. 5) confirmed the presence of d-arabinose, d-xylose
tose sugars such as d-ribose, d-arabinose, d-xylose and as pentose sugar and d-mannose, d-galactose, d-glucose
three hexose sugars such as d-mannose, d-galactose and as hexose sugar with the molecular ratio of 2:1: 2:37:58
d-glucose in TAE (Fig. 4). Importantly, the molecular respectively (Table 3).

Fig. 3 GC spectrum of standard sugar mixture (STD), TAE and DAE containing ribose (1), arabinose (2), xylose (3), inositol (4), mannose (5), glucose
(6), galactose (7)
Basu et al. Chemistry Central Journal (2017) 11:118 Page 5 of 10

Fig. 4 Mass fragmentation of TAE: 1,2,3,4,5-penta-O-acetyl ribitol (a); 1,2,3,4,5-penta O-acetyl arabinitol (b); 1,2,3,4,5-penta-O-acetyl xylitol (c);
1,2,3,4,5,6-hexa-O-acetyl mannitol (d); 1,2,3,4,5,6-hexa-O-acetyl glucitol (e); 1,2,3,4,5,6-hexa-O-acetyl galactitol (f)

Table 2 Molecular ratio of monosaccharide mixture from water soluble polysaccharides

Sample name % of CHO Compound presents # (mol%)
Arabinose Ribose Xylose Mannose Galactose Glucose

TAE 70.05 6 7 3 38.2 35.1 10.5

DAE 70.09 2 – 1 2 37 58

The functional group of purified sample was deter- presence of characteristic C=O stretching frequency
mined by FTIR spectroscopy using KBr pellet method of the carboxylic acid group. However, no such peak
(Fig. 6). The purified polysaccharides were ground with was present in TAE indicating the absence of –COOH
KBr powder and then pressed into pellets for FTIR meas- group in it. It also shows absorption bands at 1633 and
urement in the frequency range of 4000–500 cm−1. Dried 1564 cm−1 for carbonyl stretching vibration (amide-I) or
TAE and DAE (water extract/methanol extract) showed C=C groups/aromatic rings, N–H stretching vibration
a peak at 3317 cm−1 due to the stretching of the N–H (amide-II), respectively for amide linkages of the proteins
bond of amino groups and indicative of bonded hydroxyl present in it, while medium broad band at 1400 cm−1 is
(–OH) groups alcohols and phenolic compounds. The the C–N stretching mode of aromatic amine group [16].
absorption bands at 2926 cm−1 that appeared in spec- The absorption band at 989 cm−1 was due to the C–O–C
trums were due to the stretching frequency of –CH3 vibrations of proteins/polysaccharides present in the
groups. A peak at 1733 cm−1 in dried DAE indicated the extract [17]. The band at 840 cm−1 was assigned due to
Basu et al. Chemistry Central Journal (2017) 11:118 Page 6 of 10

Fig. 5 Mass fragmentation of DAE: 1,2,3,4,5-penta-O-acetyl ribitol (a); 1,2,3,4,5-penta O-acetyl arabinitol (b); 1,2,3,4,5-penta-O-acetyl xylitol (c);
1,2,3,4,5,6-hexa-O-acetyl mannitol (d); 1,2,3,4,5,6-hexa-O-acetyl glucitol (e); 1,2,3,4,5,6-hexa-O-acetyl galactitol (f)

Table 3 Elemental study of purified extracts by EDX

Element Sample C O Mg P S Cl K Ca Sn

Weight% TAE 45.67 46.76 0.54 0.30 0.27 0.91 3.67 1.35 0.53
DAE 55.54 41.40 – 0.30 – 0.18 2.57 – –
Atomic% TAE 54.93 42.22 0.32 0.14 0.12 0.37 1.36 0.49 0.06
DAE 63.41 35.49 – 0.13 – 0.07 0.90 – –

the in plane and out of plane bending for benzene ring. estimated that (Fig. 8) carbon, oxygen, magnesium, phos-
This indicated that extracts are surrounded by some pro- phorus, sulfur, chlorine, potassium, calcium, and tin are
teins and metabolites such as alkaloids and terpenoids the elements mainly found in both extract (Table 3).
that have functional groups of amines, alcohols, ketones, XRD analysis of samples represented the amorphous
aldehydes, and carboxylic acids. structure of the samples. As shown in Fig. 9, samples
The morphology of the samples was observed under showed large hump distributed in a wide range (2θ)
scanning electron microscope (SEM) with an accelerated instead of high intensity narrower peaks with interplan-
voltage of 15 kV. SEM analyses showed that the purified ner spacing (d-spacing) of 3.99 and 3.88 revealing the
samples were compact in nature with an uneven surface amorphous structure of the samples.
(Fig. 7). Also, it was not porous as observed in the sam- Thermal gravimetric analysis or (TGA) measured
ples extracted from TAE and DAE. The elemental analysis changes in weight in relation to changes in temperature.
Basu et al. Chemistry Central Journal (2017) 11:118 Page 7 of 10

Fig. 6 FTIR spectroscopy of purified sample

Fig. 7 SEM images of TAE (A) and DAE (B) purified fraction

Fig. 8 EDX of purified TAE (a) and DAE (b)

TGA is commonly used to determine selected charac- (Fig. 10). A total of 10.04 and 10.07% of weight loss for
teristics of materials that exhibit either mass loss or gain the first and 75.63 and 74.10% for the second step were
due to decomposition, oxidation, or loss of volatiles e.g. recorded for samples TAE and DAE respectively. As dis-
moisture. The TGA analysis showed degradation of TAE cussed, weight loss during the first step below 100 °C
and DAE in two well-defined steps for both materials indicated removal of moisture. From the results of DTA it
Basu et al. Chemistry Central Journal (2017) 11:118 Page 8 of 10

Fig. 9 XRD profile of T. ammi (Linn) Sprague and D. biflorus Linn purified fraction of water extract

Fig. 10 TGA and DTA response of (a) T. ammi and (b) D. biflorus purified extracts

can be clearly noticed that all the reactions were exother- it showed 3.7 cm zone of inhibition. On the other hand,
mic in nature. TAE showed zone of inhibition as 1.8 and 1.7 cm against
Importantly it was detected that TAE and DAE have Bacillus subtilis whereas, DAE showed 1.4 cm zone of
antibacterial activity (Fig. 11) against gram positive bac- inhibition. Extracts of both TAE and DAE showed sig-
teria such as Staphylococcus aureus, Bacillus subtilis. In nificant antimicrobial activity with comparison to posi-
well diffusion method, TAE showed a maximum zone tive control (tetracycline) which showed 2.9 cm zone of
of inhibition against Staphylococcus aureus (1.9 cm) inhibition. The zones of inhibition (cm) (Fig. 12) were
whereas DAE showed 1 cm zone of inhibition. A positive reported in Table 4.
control (tetracycline) was used for comparative data and
Basu et al. Chemistry Central Journal (2017) 11:118 Page 9 of 10

Table 4 Antimicrobial activity of purified extracts

Microorganism Zone of inhibition (cm)
Control TAE DAE

Staphylococcus aureus 3.7 ± 0.12 1.9 ± 0.1 1 ± 0.08

1.9 ± 0.14 1 ± 0.14
Bacillus subtilis 2.9 ± 0.11 1.8 ± 0.14 1.4 ± 0.15
1.7 ± 0.11 1.4 ± 0.13
Escherichia coli 4 ± 0.14 1.4 ± 0.13 1.2 ± 0.13
Fig. 11 Antimicrobial activity against human pathogenic bacteria of 1.4 ± 0.12 1.2 ± 0.07
TAE and DAE in comparison with positive control as tetracycline Pseudomonas aeruginosa 3.7 ± 0.14 2.1 ± 0.12 1.4 ± 0.14
2.1 ± 0.09 1.4 ± 0.11

The above results were very encouraging and deserved ± Standard deviation

further attention. It was gratifying to note that TAE and

DAE also have antibacterial activity against Gram nega-
tive bacteria such as Escherichia coli and Pseudomonas In this paper, TAE had shown better activity compared
aeruginosa. TAE showed a zone of inhibition as 1.4 cm to DAE against gram positive bacteria (zone of inhibition
and DAE showed 1.2 cm against Escherichia coli. Thus, in cm, Table 4). The percentage of aldehyde group (70%)
the extracts of both T. ammi and D. biflorus showed seemed to have no effects on the antimicrobial properties
antimicrobial activity in comparison to positive con- of the extracts. The TAE extracts had more mannose than
trol (tetracycline) which showed 4 cm zone of inhibi- glucose. The DAE extracts had shown more or less equal
tion. TAE showed the highest zone of inhibition against activity against gram positive and gram negative bacteria.
Pseudomonas aeruginosa (2.1 cm) among rest of micro- However, the DAE extracts had more glucose than man-
organisms tested. DAE showed 1.4 cm zone of inhibition nose. In both of these extracts, it seemed galactose has no
in comparison to positive control (tetracycline) which role. Glucose and mannose are epimeric. Ribose was pre-
showed 3.5 cm zone inhibition. The molecular ratios of sent in the TAE extracts. However, no ribose was seen in
monosaccharide mixtures present in water soluble poly- the DAE extracts. In this study, we observe that extracts
saccharide had significant effects on microbial activities. of TAE showed significant antimicrobial activity. The

Fig. 12 Antimicrobial activity against [I] S. aureus of TAE (A, C) and DAE (B, D); [II] B.subtilis of TAE (A, C) and DAE (B, D); [III] E. coli of TAE (A, C) DAE (B,
D); [IV] P. aureginosa of TAE (A, C) DAE (B, D) in comparison with (P) positive control as tetracycline (N) negative control as normal saline water
Basu et al. Chemistry Central Journal (2017) 11:118 Page 10 of 10

results of antimicrobial activity of these polysaccharides Publisher’s Note

as demonstrated herein are new and novel. The effects Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations.
polysaccharides against human sugar levels are studied
by some authors. But those polysaccharides (TAE and Received: 9 August 2017 Accepted: 10 November 2017
DAE) can also act as promising antimicrobial agents are
not explored.

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Competing interests
The authors declare that they have no competing interests.