WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

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Volume 3, Issue 9, 592-603. Research Article ISSN 2278 – 4357

CHARACTERISATION OF Solanum diphyllum L. - A MEDICINALLY

POTENTIAL SPECIES FROM SOUTHERN WESTERN GHATS IN
KERALA USING SEM AND FTIR SPECTROSCOPY

V.S.Anilkumar1, P.J. Aswathy2, A V Sunila3 & K.Murugan3*

1
Department of Botany, Government College, Kottayam, 686013 Kerala, India
2
,Department of Botany, Government College For Women, Thiruvananthapuram,
695 014 Kerala, India
3
Plant biochemistry and molecular biology laboratory, University college,Thiruvananthapuram
695 034, Kerala, India.

ABSTRACT
Article Received on
26 June 2014, Solanum diphyllum L., a medicinal herb, native of Mexico and Central
Revised on 16 July
2014, America grows wildly as naturalized in many tropical and subtropical
Accepted on 07 August 2014
countries. Scientific data addressing its micromorphological features
are unavailable. The plant showed promising cytotoxicity against colon
*Correspondence for Author and breast carcinoma cell lines, and could be considered as a potent
Dr. Murugan K
source of anticancer molecules. The present research is aimed at
Plant biochemistry and
investigating ultrastructural features of leaf, pollen and seed sporoderm
molecular biology laboratory,
University college, using scanning electron microscopy and also functional groups by FT-
Thiruvananthapuram Kerala, IR spectroscopy. Leaf epidermis shows glandular and non-glandular
India. hairs. Stomata are anisocytic stomata. Presence of a thick waxy layer
over the leaf surface protects the leaves from desiccation. Pollen,
trichomes and seed texture patterns were analysed and the data compiled for species
discrimination. The morphology of the spermoderm as well as pollen were found to be useful
as taxonomic tools for species level identification as well as comparison as these features are
genetically determined The spermoderm shows a retticulate pattern with broad lumen and
compact hilum. Pollen grains were trizonocolporate and prolate spheroidal in shape. FTIR
spectral data reveals specific absorption peaks for the species. Peaks at 1041.56, 2360.87,
3118.9, 3736.12, 3790.12, 3803.63, 3824.84 and 3838.34 are characteristic to Solanum
diphyllum.. FTIR spectral data was further analysed with a view to distinguish the functional

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chemical groups in the dried leaf tissue which in turn provides relevant leads for exploring
the pharmacobotanical potentials of the species.

KEYWORDS: Solanum, Scanning Electron Microscopy, FTIR spectrum.

INTRODUCTION
Solanum is the largest genus in Solanaceae and represents one of the 10 most species rich
genera of angiosperms [1]. The genus is mainly tropical but few are temperate and consists of
[2] [3]
over 2000 species distributed worldwide . They include common source of vegetables ,
[4]
noxious weeds and medicinal herbs and contain unique alkaloids and other biochemical
constituents used for the treatment of diverse ailments (diabetes, cholera, bronchitis, high
blood pressure) and as laxatives [5]. In spite of the economic and medicinal value of Solanum
species, no serious attention has been paid to the diversity, characterization and taxonomical
identification at the biosystematic level. This often proves to be helpful as there is great
degree of adulteration practices with allied species of the medicinally potential forms. The
selected species viz. Solanum diphyllum is naturalised in Kerala. Hossain et al. [6] studied the
phenolic content, anti oxidative, anti alpha amylase and anti alpha glucosidase activities of
the fruits, leaves, stem and roots of S. diphyllum and suggested that this herb can be used
against cancer, diabetes and inflammations. Usually the plants are collected from the wild
and brought to the processing centres or traditional selling outlets as such in dried form and
hence sometimes it is very difficult to assess the authenticity of these medicinal herbs. The
habit is shruby, minutely pubescent, stem terete, leaves 2 at each node, unequal, inflorescence
racemose fascicle, calyx cupular, 5-lobed; lobes broadly triangular, corolla stellate, creamy
white, 5-lobed, incurved, stamens 5, epipetalous, berries globose, slightly constricted in
middle, glabrous, yellow or reddish yellow; seeds reniform or discoid, thickened at margins
and yellow in colour. In this juncture, present study aims in analysing the micro
morphological features of the species using SEM, coupled with FTIR spectral analysis of the
dried plant tissue that provides species specific information, helping to delineate the species
from allied forms.

MATERIALS AND METHODS

Plant material
Fresh specimens of pollen, seed and leaves of the specimen were collected from
Thiruvananthapuram and Wayanad districts of Kerala, India and were identified by

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comparison with the voucher specimen from Kerala Forest Research institute (KFRI, Kerala).
The voucher specimens were deposited at the herbarium of Jawaharlal Nehru Tropical
Botanic Garden and Research Institute, Palode. Five accessions were analyzed in the present
study.

Scanning electron microscopy (SEM)

The leaf, pollen and seed surface architecture of the accessions were analyzed using scanning
electron microscopy (SEM).

Leaf: Fresh leaf pieces (10 x 10 mm2) from the studied species were immersed in a fixative
solution of 3% glutaraldehyde in 0.1 M phosphate buffer for 24 h. Samples were washed for
15-30 min with the buffer and dehydrated in graded ethanol series. Samples were then
critical-point dried using CO2, sputter coated with gold under vacuum and viewed with
Hitachi (S-450) scanning electron microscope operating at 15 kV. Images were captured
digitally with an Image Slave computer programme for Windows.

Seeds: Mature dry seeds (without fixation) were glued to aluminium stubs and coated with
gold-palladium to a thickness of 40-50 nm using a JEOL Finecoat Ion Sputter JFL 1100. The
specimens were viewed in a JEOL scanning electron microscope (JEOL JSM T220A, Japan)
and photographed at different magnifications. For each accession, at least 15 seeds were
randomly selected and studied.

Pollen grains: Anthers separated from 70% alcohol fixed flower buds are used for pollen
collection. Acetolysed pollen was attached to stubs with double-faced carbon tape. The stubs
are gold-coated in sputter coater for one minute and examined. The terminology used is in
accordance with Punt [7].

FTIR spectroscopy
The leaves (approximately 3-4 cm) were taken from different plants and were pooled as one
sample. Then the samples were dried in shade till the tissue becomes fully dry and crispy.
Tablets for FTIR spectroscopy were prepared in an agate mortars by mixing leaves powder (2
mg) with KBr (1:100 p/p). The absorbance spectra were measured between 300 and 4500 cm-
1
. At least three leaves were collected and minimum three spectra were obtained from each
sample.

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A FTIR spectrometer (FTIR-NEXUS 670, Thermo Nicolet Corporation, America) was used.
Spectra were obtained in 32 scans co-added, 4000 resolution and 2.0 gains. The parameters
for the Fourier self-deconvolution were a smoothing factor of 15.0 and a width factor of 30.0
cm-1. De-convolved and second-derivative spectra were calculated for Fourier self-
deconvolution and the bands were selected and normalized to unity with Omnic 7 software.
Curve-fitting of the original spectra was performed with Origin 7 software. The band position
of functional groups was monitored with Knowitall 7.8 software. The spectral region between
3000 and 2800 cm-1 was selected to analyze lipids. The spectral region between 1800 and
1500 cm-1 was selected to analyze proteins. The spectral region between 1200 and 1000 cm -1
was selected to analyze carbohydrates.

RESULTS AND DISCUSSION

Leaf
The taxonomic value of epidermal morphology is well documented in botanical literature
[8,9],
In comparative investigations of angiosperms, trichomes were considered to have great
importance. The size of trichomes range from a few microns to several centimeters and they
[10]
exhibit tremendous species specific diversity in shape and hence often used as diagnostic
characteristics for the identification of plant species [11].

The lamina of S. diphyllum L. is dorsiventral. Both surfaces of the leaf samples i.e., adaxial
and abaxial surfaces were analyzed using SEM. In Solanum diphyllum, the adaxial leaf
surface displays more or less orderly packed cells. Leaf epidermis has few sparse trichomes,
simple unicellular and non-glandular and multicellular glandular hairs which consisted of a
short multicellular stalk and unicellular secretory head at the top round to oval in shape.
Glandular trichomes were more in young leaves. Stomata were anisocytic i.e. stomata are
surrounded by three cells, one of them is distinctly smaller than the other two. The present
[12] [13]
results agrees with Metcalfe and Chalk (1950), Ahmad (1964), and Dwelle et al. [14]
[15]
(1983), Murthy and Inamdar (1980) who worked on other Solanum species and recorded
the presence of cruciferous types i.e. three subsidiary cells per stoma. In other words, on the
[16]
dorsiventral leaf, anisocytic stomata were confined to one surface (Watson and Dallwitz,
1992) and simple uniseriate or glandular hairs (Edmonds, 1972) [17] were the characters that
have been detected and shared with other members of Solanaceae and genus Solanum.
Hamada et al. [18] also reported similar morphological features in this species from Egypt. The
stomatal openings were ovate-rounded shaped. The electron microscope scanning for the leaf

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showed that the epidermis is sculptured by islands of rounded crystalloids epicuticular wax.
The stomata were not present on the same level. As a result of wax elevation, stomata were
raised, slightly depressed or depressed due to the presence of the rims, which give the shape
of depressed stomata. The stomata were sparse on the upper leaf surface and their frequency
was further reduced on the lower leaf face. Both types of trichomes such as glandular and
non-glandular were sparsely distributed (Fig. 1A). Along the margins, single celled non-
glandular trichomes with blunt tips were distributed (Fig.1B). Similar trichomes were also
noticed along the veins (Fig.1C). The glandular trichomes were short stalked with
[19]
multicellular heads and randomly distributed (Fig. 1D). According to Wagner et al. , non
glandular trichomes are present in most angiosperms but also could be seen in some
[20]
gymnosperms and bryophytes. In contrast, Fahn reported that glandular trichomes are
usually multicellular and found to occur in approximately 30% of vascular plants. They have
the capacity to produce, store and secrete large amounts of different classes of secondary
metabolites.

The distinct foliar features of the species can be used as one of the taxonomic identities.
Despite this, hairs on leaves have other functional roles. Trichomes constitute a mechanical
[21]
barrier that hinders insect movement and/ or feeding and chemical compounds in
[22]
glandular trichomes can be deterrent or toxic to several herbivores . These glands contain
important secondary metabolites including terpenes, essential oils, flavonoids and lipophilic
components [23]. In most species the source of these secondary metabolites has been attributed
[22]
to the trichomes . The possession of glandular trichomes is characteristic of the genus
Solanum and of many other members of Solanaceae with the exception of Nicotiana glauca
[24].
and Solandra nitida Anil kumar and Murugan (2013) reported the trichome phenotypes
and their distribution in various Solanum species from Kerala which explains the diversity of
[25]
foliar hairs in the genus . The foliar micromorphology of the studied species thus gives
information regarding their adaptation to environmental stresses apart from their role in
taxonomic realm.

In Solanum diphyllum, non glandular trichomes are distributed along the leaf margins in low
density and very randomly on the leaf blade. This scarcity of trichomes can be correlated with
the very low stomatal frequency in the leaf epidermises of the species. Presence of a thick
cuticular layer over the leaf surface may presumably be compensating for the bioshield of
[26]
trichome mat that characterise the spiny Solanums. Ludlow and Muchow reported that

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this feature of waxy bloom on leaves helps with maintenance of high tissue water potential
and is therefore considered as a desirable trait for drought tolerance.

Figure 1: SEM image of the upper leaf surface of Solanum diphyllum L.

A- Showing sparse distribution of trichomes and orderly arranged epidermal cells (Tr-
Trichome). B- Short single celled non glandular trichomes with blunt tip along the margin. C-
Leaf surface showing the both single celled nonglandular trichomes along the veins (Tr-
Trichome). D- Lower leaf surface with short glandular trichome and stomata (Gt-Glandular
trichome,St-Stomata).

Seeds
Seed morphology has been shown to provide useful characters for the analysis of taxonomic
[27,28,29,30].
relationships in wide variety of plant families . In addition to gross morphology of
seeds, the details of the sculpturing of the outer seed coat can be quite variable and of
systematic importance. Recently, the systematic implication of seed coat diversity has been
carried out in Ipomoea [31], Minuartia [33] and Lepidium [32]. The seeds of Solanum diphyllum
are more or less reniform in outline (2.21 x 2.73 mm) (Fig. 2 A). The spermoderm shows a
retticulate pattern (Fig.2B) in which the lumens range in dimensions from 65.9 µm x 121 µm
and 62.2 µm x 75.1 µm. The lumens are found to be comparatively larger towards the
periphery of the seed and the proper hilum region seems to be more or less compact (Fig.

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2C). Testal cells are not markedly isodiametric and the cell walls are obviously sinuate. The
lumen is deep viz. the lateral anticlinal cell walls are longer, thin and gradually tapering
rather being rounded at the top. These anticlinal lateral testal walls are not straight and
smooth but shows an irregular sinuately thickened ornamentation pattern (Fig. 2D). The distal
appendages are represented as a minute ribbon at the summit of the broadly thickened part of
the wall except towards the periphery of the seed, where flap like projections with
differentiated finger like laciniations could be seen (Fig 2E). All these features are of
particular systematic relevance that delineates the species. In other taxa also, the spermodem
sculpturing patterns have been found to be different in different species of the same genus
[33]
and hence of taxonomic importance . Considerable differences were also observed in the
seed coat morphology both among and within the genera in the tribe Hyoscyameae of
Solanaceae[34].

Figure 2: Spermoderm of Solanum diphyllum L. under SEM.

A- Whole seed surface. B- Enlarged view showing lumens. C- Distal ends of anticlinal walls
showing the presence of thin thread like ornamentations. D- Hilum region of the seed
showing slightly elongated lumens towards the hilum and compactness of the proper hilum
part.

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Pollen grains
Pollen characters have received more attention in taxonomic and pollen morphology.
However, the ultra-structure of the pollen wall, its stratification and internal structure can
[35]
hardly be studied by light microscope . Therefore Scanning and Transmission Electron
[36]
microscopy become necessary in examining these characters, El- Ghazaly and Harky et
al.[37] reported the morphology of pollen grains of many plant species. So far, no previous
ultra-structural pollen studies have been reported for Solanum diphyllum. Pollen grains were
trizonocolporate and prolate spheroidal in shape. The grains are spherical in equatorial view
measuring E 11.1 µm x 11.1 µm/ P 11.4 µm x 11.4 µm. In polar view, they are convex with
obtuse angles (3A). The exine ornamentation is scabrate. In equatorial areas the sexine is
thickened to form a margo. The colpus is broader, relatively long extending nearly to the
poles and having ornamentations. Prominent finger like ridges can be observed (Fig.3B).
Interestingly, some grains showed the fusion of two colpi as observed in their polar view
(Fig.3C).

Figure 3: SEM analysis of the pollen grains of Solanum diphyllum L.

A- Equatorial view of the pollen showing the colpus and ridges. B- Polar view of the pollen.
C- Apocolpial view showing union of two colpi (Fc-Fused colpi).

FTIR spectroscopy
Fourier Transform Infrared (FTIR) Spectroscopy is a rapid, non-invasive, high-resolution
analytical tool for identifying types of chemical bonds in a molecule by producing an infrared
absorption spectrum that is like a molecular “fingerprint”. In the present investigation, FTIR
spectral data reveals specific absorption peaks for the species. Peaks at 1041.56, 2360.87,
3118.9, 3736.12, 3790.12, 3803.63, 3824.84 and 3838.34 are characteristic to Solanum
diphyllum (Fig 4). The peak at 1041 corresponds to aliphatic amines, 2360 to nitriles, 3118 to
aromatic alkenes while peaks in the 3700 range specifically corresponds to N-H stretch amide

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groups. This species is found to have higher concentrations of nitro compounds and aromatics
in the peak range 1550 and 1600. Several studies regarding the applicability of FTIR spectral
[38,39,40]
data in the analysis of the bioactive compounds of plants have been conducted .
[41]
Recently, Zimmermann and Kohler employed IR spectroscopy of pollen to identify
species, genus and environmental conditions. This preliminary spectral analysis reveals the
fundamental phytochemical profile of Solanum diphyllum. IR finger printing of various
[42]
Solanum species of Kerala has already been conducted in a taxonomic perspective . The
present investigation provides the first report of its kind for Solanum diphyllum.

Figure 4: FTIR Spectrum of Solanum diphyllum L.

CONCLUSION
The study persuades the applicability of SEM and FTIR spectroscopy in unveiling the species
specific traits of Solanum diphyllum that can serve as marker characters. Since the pollen and
seed features are genetically fixed traits, they can obviously be considered as marker features
of a plant species. The FTIR spectral data was found to be useful in determining the
phytochemically relevant functional groups so that the pharmacological potential of the plant
species can be identified and further exploited. Similar approaches are warranted for other
commercial medicinal herbs that are being widely used across the globe.

ACKNOWLEDGEMENTS
The authors are thankful to The Director, National Institute of Interdisciplinary Science and
Technology( NIIST), Thiruvananthapuram, Kerala for providing SEM facilities, Dr. A. G.
Pandurangan (Scientist F, Jawaharlal Nehru Tropical Botanic Garden and Research Institute,
Palode, Thiruvananthapuram, Kerala, India), for authentication of the samples and the

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Director, JNTBGRI (Palode, Thiruvananthapuram, Kerala, India) for providing the facilities
for the deposition of herbarium specimens at the Institute herbarium.

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