macro, micro-morphological and bioactivity aspects of naturalized exotic solanum diphyllum l
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Al-Azhar Bull. Sci. (ISCAZ 2010). (March, 2010): pp. 175-206.MACRO, MICRO-MORPHOLOGICAL AND BIOACTIVITY ASPECTS OF
NATURALIZED EXOTICSOLANUM DIPHYLLUML.
FATMA A. HAMADA, ARAFA I. HAMED*, MOHAMED G. SHEDED ANDABDEL SAMAI M. SHAHEEN
South Valley University, Faculty of Science, Botany Department, Egypt.*To whom correspondence should be addressed.
Abstract
Solanum diphyllum L. is a native plant to Mexico and Central America, and distributed in
many places around the world. Currently escaped from cultivation as an ornamental plant andgrows wildly as a naturalized exotic plant in some places in the Egyptian Nile Valley region.
Solanum diphyllum L. morphology and anatomy showed similarities with its close affinities in
its genus. The presence of lenticel-like structures may be a response to the plant defence
needs and/or may be a result of an adaptation to its surrounding environment; in which the
plant discard excess storage materials to decrease the effect of stored osmolytes, and the
presence of epidermal wax and tannins may help the plant to acclimate to the surrounding
light intensities. Solanum diphyllum L. showed a promising cytotoxisity against colon (HCT
116) and breast (MCF7) carcinoma cell lines, and could be considered as a potent source of
anticancer compounds. The plant might represent a good model in terms of morphological,
anatomical and physiological adaptation to its surrounding environment; revealing a hidden
symphony.
Key words: Solanum diphyllum L.; anatomy; tannin sacs, lenticels; cytotoxicity.
Introduction
The Solanaceae contains between 3,000 and 4,000 species in about 90 genera
(Knapp et al. 2004). The members of the Solanaceae have a very important
relationship with human being as some are noxious weeds, while others are of a
great economic importance as a source of food; e.g. Solanum tuberosum L. (potato),
Lycopersicum esculentum Mill. (tomato), Nicotiana tabacum L. (tobacco), andCapsicum frutescens L. (chilies), ornamentals (Cestrum spp., Petunia hybridia and
Solanum spp.), and as an extremely important drug sources in medicinal
pharmacology and drug therapy (e.g. Hyoscyamus, Datura and Belladona). The
Genus Solanum L., has a world-wide distribution, and is one of the largest genera of
the flowering plants; is considered as the largest and the most diverse genus in the
Solanaceae, with 1500-1700 species, and is one of the ten most species-rich genera
of flowering plants (Mabberly, 1997; Frodin, 2004).
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FATMA A. HAMADA, et. al176Boulos (2009) reported that the Solanaceae is represented in Egypt by eight
genera and 30 species; nine of them are related to genus Solanum. This poor
representation of wild and naturalized species is compensated by relatively large
number of introduced and cultivated 42 species (Hepper, 1998).
Solanum diphyllum L. is an attractive shrub, has tiny white flowers and bright
orange fruits. It has been widely cultivated in tropical and subtropical areas where it
escaped in many of these regions, e.g.; Florida, Java, the Antilles, Southern France,
Italy, Philippines and China. This plant is usually widespread in drier habitats
(D'Arcy, 1974; Knapp, 2002). In Egypt the plant is spread in Aswan and Giza
governorates (Figure, 1).
Figure (1): (A) Solanum diphyllum L. distribution in the world. (B) Its distribution in
Egypt.
Solanum diphyllum L. belongs to genus Solanum section Geminata (G. Don)
Walp. (Knapp, 2002). Solanum diphyllum L. has been recorded as a new record for
the Egyptian flora by Shaheen et al. (2004).
It has the following common and local names; twinleaf nightshade, two-leaf
nightshade, tomatillo (Mexico) (Knapp, 2002), huang guo long kui (China) (Cheng-
yi and Raven, 1994) and amatillo (FDT, 1995).
**
A B
*
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MACRO, MICRO-MORPHOLOGICAL AND BIOACTIVITY. 177
The relationship between the plant structure and its environment is one of the
most attractive subjects; it is of great interest to plant anatomists and taxonomists at
the morphological level, or even at the physio-morphological level.
No one can deny that nature has provided many effective anticancer agents
which are currently in use, some derived from microorganisms such as dactinomycin
and doxorubicin, or from plants in the case of vinblastine, irinotecan, topotecan,
vincristine, taxanes, etc. (Ruffa et al., 2002). Despite growing research on flora, only
ten percent of the approximately 250 000 species of the higher plants is estimated to
have been chemically and pharmacologically investigated. The potential of many
plants remains virtually untapped and could be studied (Cragg and Newman, 1999).Continuing efforts of scientists around the world are not stop to seek bioactive
components and new cytotoxic agents from natural and traditional herbal sources.
As interest in herbal medicine and herbal anticancer therapy in particular has
increased, one of the main goals of anticancer therapy and prevention is the
discovery of compounds that are relatively potent and selective to tumor cells and
less toxic for the normal cells.
To our knowledge, little is known about the morphological and anatomical
characteristics of Solanum diphyllum L.. So, one of the objectives of the currentresearch is to reveal the most important morphological and anatomical features of
the Solanum diphyllum L. Moreover, the present work investigates the relationship
between its morphological and anatomical characters, its chemistry and its
surrounding environment. Also, the work aims to evaluate the anticancer bioactivity
via testing the in vitro cytotoxicity of the methanolic extracts of different parts of
Solanum diphyllum L. against three different cell lines of common malignant tumors
in Egypt and in many developing countries.
Experimental
The Identification and world distribution of the collected Solanum diphyllum L.
specimens were insured through the Missouri botanical garden web site, New York
botanical garden web site and Aluka digital herbarium web site. The plant specimens
were compared with a loaned herbarium sheet from Kew botanical garden. The
specimens were collected from Giza and Aswan governorates confirming the
presence of Solanum diphyllum L. as a naturalized exotic species in the Egyptian
Nile Valley area. A detailed list of specimens including herbarium voucher
references can be obtained from the authors.
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FATMA A. HAMADA, et. al178A. Morphological investigation:
Specimens' surface patterns were first investigated by the 7-14 magnification
using an Olympus stereomicroscope. Measurements for length, width and thickness
were done for 15 replicates using a ruler, a microscopic micro-ocular lens, and a
digital caliper.
B. Anatomical investigation:
Cross-sections were performed on fresh samples of stems, leaves, petioles and
roots; replicates of four. Plant specimens were fixed, preserved, and stained
according to Alsahar and Nassar (1998). The slides were examined and
photographed using Bresser Biolux Al 20x-1280x light microscope Germany, LCD
Micro Bresser 40x-1600x light microscope Germany, and Leica DMLB microscope
Germany; coupled to JVC TK 1380 Japan, still digital camera.
C. Scanning microscope investigation:
Leaf and stem specimens were fixed in 10% formalin, and dehydrated in cold
ethanol series (National Research Centre technician, person. comm.). All tissues
were sputter-coated with 15 nm of gold-palladium and viewed with a JEOL-JXA-
840A electron probe micro-analyzer, Japan.
Analysis of certain metabolites of the Solanum diphyllum L. leaf, stem and root:
1. Total tannins were estimated using the gravimetric method (Copper acetate
method) according to Ali et al. (1991).
2. Total carbohydrates were determined spectrophotometrically (Cherry,
1973).
3. Total flavonoids were determined spectrophotometrically (Karawya and
Aboutable, 1982).
4. Total alkaloids were estimated using the gravimetric method according toBalbaa (1986).
5. Total saponins were determined colorimetrically (Honerlogen and Tretter,
1979).
In vitro cytotoxisity essay experimental:
Plant material: Different plant parts ofSolanum diphyllum L.were collected from
Aswan, Egypt, in February 2005. A voucher specimen (No. 10.520) was retained in
the Botany Department Herbarium, Faculty of Science at Aswan, Egypt.
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MACRO, MICRO-MORPHOLOGICAL AND BIOACTIVITY. 179
Extraction and isolation:
A. Shoot extract:
The dried and powdered aerial parts of stem and leaves ofSolanum diphyllum L.
(321 gm) were exhaustively extracted with MeOH: H2O (8: 2) using Soxhlet
apparatus. The alcoholic extract was condensed to syrupy consistency (54 gm).
B. Root extract:
The dried and powdered roots (76 gm) were exhaustively extracted with MeOH:
H2O (80%) using Soxhlet apparatus. The alcoholic fraction was condensed to syrupy
consistency (8 gm).C. Fruit extract:
The dried and powdered mature yellow-green fruits (54 gm) were exhaustively
extracted with MeOH: H2O (80%) using Soxhlet apparatus. The alcoholic extract
was transferred to a separating funnel and was shaken with hexane till exhaustion.
The defatted alcoholic fraction was condensed to syrupy consistency (12 gm).
In vitro cytotoxic acitvity assay:(cell survival test; growth assay and viability).
The cytotoxic activities of the methanolic extracts of different parts ofSolanum
diphyllum L. were studied against three human tumor cell lines; HCT116 (colon
carcinoma), MFC7 (breast carcinoma), HEPG2 (liver carcinoma), and normal skin
melanocytes cell line (HFB4). The cell lines were obtained from (ATCC, Mo, USA)
culture collection, they were maintained by serial sub-culturing. The cytotoxicity of
the tested crude extracts was carried out by a colorimetric assay using
sulforhodamine- B (SRB) dye (Skehan et al., 1990) at Pharmacology unit, Cancer
biology department, National Cancer Institute, Cairo University.
Briefly, the cells were seeded into a 96-well microtiter plate at a cell density of
(5 x 103
cells/well). After 24 hrs incubation, the monolayer cells were treated withvarious concentrations (0, 5, 12.5, 25, 50 g/ml) of different crude extracts, in which
different crude extracts (1mg/ml) were diluted to the required concentration using
1% DMSO, and each concentration was triplicate and the experiment was repeated
twice. After 48 hrs of incubation at 37C in 5% CO2 incubator, cells were fixed,
washed and stained with sulforhodamine-B (SRB) stain, excess stain was washed
with acetic acid and attached stain was recovered with Tris EDTA buffer. The
optical density (O.D) of each well was measured spectophotometrically at 570 nm
using ELISA microplate reader (Sunrise Tecan, Tokyo, Japan).
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FATMA A. HAMADA, et. al180The cell survival was calculated as follows: survival fraction = O.D (treated
cells)/ O.D (control cells). The relation between surviving fraction and each crude
extract concentration was plotted to get the survival curve of each tumor cell line
and the IC50 (inhibitory concentration 50%) of each extract was calculated using
statistical computer program PRISM 5 (Sato and Kameya, 2001).
Statistical analysis:
Investigated parameters were subjected to analysis of variance (ANOVA)
performed using SPSS 11.0 for Windows (SPSS, Inc., Chicago, IL, USA). Values
with p < 0.05 were considered significant. The results were expressed as mean
S.E. The significant differences among means were compared by LSDtest at level0.05 and 95% confidence intervals. Charts were performed using Microsoft Excel
software.
Results and discussion
Morphological description:
Small perennial shrubs 0.5-1.5 m tall occasionally reach 2m (Figure, 2, A), root
is a normal woody tap root, 10-30 cm length, 0.5-2 cm diameter, gives underground
reproductive structures forming underground root laterals (Figure, 3). Upright
woody stem green to brown green terete with 2-3 ridges when young, terete
lenticellated pale brown when old; minutely puberulent with minute uniseriate
eglandular trichomes 0.01-0.05 mm long, glandular hairs may present on young
branches.Internodes 1-3 cm, branches and main axis thickness 0.2-2.5 cm, difoliate
sympodial units with geminate leaves; leaf pair is markedly anisophyllous (unequal
paired).
Leaves, oblanceolate, 10-16 x 2.5-4 cm on young non-reproductiveshoots and
lower branches, leaf margins entire; glandular and eglandular trichomes sparsely
present on the margins and on the main vein beneath, the apex acute, the baseattenuate; decurrent on the petiole and stem, petiole 2.5-10 mm long. leaves on the
reproductive shoots; major leaves elliptic to oblong, geminate, widest at the
middle; 5-9 x 2.5-3 cm, the apex rounded, short acuminate or acute, the base acute
to attenuate, decurrent on the stem and on the petiole, petioles 1-8 mm long; leaf
margins and the main vein beneath have sparse trichomes, minor leaves ovate to
obovate, 1-5 x 0.8-2.5 cm, the apex rounded, the base attenuate, decurrent on the
petiole; petioles 1-4 mm long (Figure 2; B). Venation is eucamptodromous in which
the secondaries veins are not terminated at the margin but turned upward, gradually
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MACRO, MICRO-MORPHOLOGICAL AND BIOACTIVITY. 181
apically diminished in size, and connected to superadjacent secondaries by a series
of cross veins.
Inflorescences opposite the leaves (leaf-opposed), short racemose, often
subumbellate, peduncle 0.7 -1.1 cm; have minutely spare trichomes, pedicel 0.75-
1.7cm long; glaborous, flowers 5-30, pentamerous; sepals 1.9-3 x 0.8-1.5 mm
deeply deltoid lobed, papillose nectaries on internal tips of the sepals.
Figure (2): A) Solanum diphyllum L. (whole plant). B) Solanum diphyllum L. (a branch).
Bar = (A = 7 cm, B = 2 cm)
Corolla 3-4 x1-2 mm, deeply lobed, white the outer tinged with lavendar in
sunny places and become obovoid directly before anthesis, papillose nectaries on
internal tips of the petals.
Stamens stout, equal, anthers 1.3-1.6 mm x 0.5-1 mm, the filaments glaborous,
0.3-0.6 x 1-7 mm. Ovary glabrous 0.8-1.1 x 0.7-1.1 mm; style glabrous 3-7 x 0.15-
0.4 mm; straight, stigma minutely papillose capitate. Fruit a bright fleshy berry,
yellow or orange globose when ripe; 0.5-1.2 cm in diameter. Seeds flattened-
reniform yellow to pale brown 2-3 x 1.5-2.5 x 0.4-0.6 mm (Length x Width x
Thickness), the surfaces minutely pitted, a single fruit contains 20-65 seeds.
The above ground part description was consistent with the description given by
Knapp, et al. (2002) with little differences that may result due to differences in the
geographical and environmental conditions. The growth ofSolanum diphyllum L. is
sympodial type; the sympodial type of growth has long been reported for Solanaceae
(Sachs, 1882; Wettstein, 1891; Solereder, 1908). As in most other Solanums in
section Geminata, flowers ofSolanum diphyllum L. are pentamerous, actinomorphic
A B
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FATMA A. HAMADA, et. al182and gamopetalous, and well-developed ovaries are globose and glabrous, seeds are
flattened-reniform typical of those found in family Solanaceae as mentioned by
Knapp (2002).
.
Figure (3): A, B Solanum diphyllum L. roots; showing main root and root laterals.
(Bar = 1 cm.)
It is well known thatthe primary role of roots in addition to providing stability isto obtain water and nutrients and in some cases work as organs for re-growth and
reproduction (Jackson, 1900). Solanum diphyllum L. has underground reproductive
root laterals. The function of these laterals is similar to that of rhizomes as they are
vegetative reproduction underground organs and carbohydrates store. This type of
vegetative propagation; to have growth from adventitious buds on roots was
recorded in other families as Podostemataceae or genera including Euphorbia
(Euphorbiaceae) and Rorippa (Brassicaceae) (Klimesova and Martinkova, 2004).
Moreover, it was well documented by Cuda et al. (2002) and by the NAPPO (2003a) for Solanum elaeagnifolium and Solanum carolinense. Moreover, Boyd and
Murray (1982)stated that Solanum elaeagnifolium can spread by root fragments
Several species in the genus Solanum showed underground reproductive
structures, which give the ability to propagate clonally from these underground
laterals (DAWM, 2006; NAPPO, 2003 b). This phenomenon was observed in arid
Solanum spp. as those plants, which have been mentioned previously. Solanum
diphyllum L. has been reported to grow in arid habitats by Knapp, 2002 and D'Arcy
ShootAnother shoot
A B
Underground lateralconnection (root lateral)
Shoot
Undergroundlateral connection
Vertical main root
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MACRO, MICRO-MORPHOLOGICAL AND BIOACTIVITY. 183
(1974), so it seems that plants that have the same morphological characters, and
grow in the same environment are taxonomically related (Dennett, 2006).
Anatomical description:
A) The Root
Secondary thickening was present, as transverse section of the root showed
periderm, secondary cortex, secondary vascular bundle, and pith. Periderm
composed of multi-layered thin walled cork cells arranged in regular radial rows.
Within and underneath the periderm, tannin sacs were detected. Followed by the
secondary cortex; starch granules were detected within these cells. The pericycle andthe endodermis were not detected.
Scattered patches of lightly lignified fibers, and mucilage idioblasts were
scattered within the cortex. Complete vascular bundle was detected, secondary
phloem externally followed by 2-3 strips of fascicular cambium. Xylem forms a
continuous cylinder, traversed by narrow rays. Vessels were very variable in size,
protoxylem strands were 2 or 4 at the center of the section arranged in loose radial
oblique rows. Parenchyma usually was scanty few limited cells; rays were
exclusively uniseriate occasionally biseriate (Figure, 4).
In general our observations are in accordance with those of Metcalfe and Chalk
(1950) detected for family Solanaceae and genus Solanum.
Figure (4): A) Solanum diphyllum L. middle-aged root B) old root (Bar = 0.05 mm.).
AB
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FATMA A. HAMADA, et. al184Thick laterals and vertical roots were found to have similar root anatomy with an
obvious secondary growth. According to Bell et al. (1996), Klimesova and
Martinkova (2004); Guerrero-Campo et al. (2006) there is a distinction between
root-sprouters and rhizomatous plants. Dicotyledonous stems whether they are
above or below the soil surface, possess dicotyledonous stem anatomy with
secondary thickening and a clearly defined vascular region, and pith in the centre of
the stem. The underground lateral connections were found to possess root rather than
stem anatomy, therefore the suggestion that the underground connections are
rhizomatous is rejected.
B) The Stem
The stem cross section outline is terete with 2-3 ridges in young stem, terete in
older one. The epidermal cells are single layered of radially elongated cells covered
by thin layer of cuticle. The epidermis has sparse of three types of trichomes;
unicellular and multicellular (2-3 cells) uniseriate non-glandular and multicellular
glandular trichomes particularly in young branches.
The epidermis replaced by the periderm in the secondary growth. Tannins
deposited in the epidermis or the periderm outer most cells as tannin sacs. Cortex
consisted of three cell types, 1-3 layers of chlorenchymatous cells followed by 3-6layers of tangentially expanded angular-lamellar collenchymatous cells, and wide
part of paranchymatous cells, some of which filled with dark material; idioblast
mucilage cells containing druses crystals. Mucilaginous idioblasts were observed;
varied from being very sparsely scattered to very dense grouped in the cells.
The vascular bundle represented by a complete cylinder of vascular tissue of bi-
collateral vascular bundle. Secondary thickening is present as secondary external
phloem and secondary xylem, which can be easily distinguished, two to three rows
of fascicular cambium in between the secondary phloem and the secondary xylem
were present (Figure, 5).
The vascular bundle showed to some extent well developed internal phloem if
compared with the external one. The outer phloem consisted of considerable number
of sieve cells that mixed with numerous parenchyma cells, with lignified scattered
fibers which may represent the pericycle. The inner phloem was noticeably wider.
Xylem showed inconsistent size of vessels, as they varied in diameter in a radial
arrangement, the small narrow primary xylem vessels were towards the inner side,
while the wide secondary vessels were towards the outside, and xylem rays were
uniseriate.
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MACRO, MICRO-MORPHOLOGICAL AND BIOACTIVITY. 185
Figure (5): Solanum diphyllum L. stem. A) Lenticels like structures have brown external
excretion on the bark (Bar = 0.3cm). B) T.S in young stem (Bar = 0.05 mm).
C) T.S in mature stem shows tannin sacs (Bar = 0.05 mm). D) T.S in the
mature stem has lenticel- like structure (Bar = 0.05 mm). E) Eglandularmulticellular uniseriate trichome, using SEM (Bar =100 m). F) Idioblast
mucilage cell with druses crystal inside (Bar = 0.05 mm).
The stem characterized by wide pith especially in young branches with relatively
thin walled polygonal parenchyma cells which increase in size towards the centre
and have crystal sands.
Between the innermost xylem elements and the pith lay the internal phloem.
Some of the pith cells were sclerosed between pith and the internal phloem. This
character was found in some species of Solanaceae e.g. Salpichroa and Sessa
B
C
D
E
F
A
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FATMA A. HAMADA, et. al186(Metcalfe and Chalk, 1950). There were other characters, which are common in
Solanaceae such as the presence of bicollateral vascular bundles (Solereder, 1908;
Fahn, 1982) and the presence of crystal sands (Metcalfe and Chalk, 1950).
In addition to lenticels that were formed as a result of the secondary growth,
there were other lenticel-like structures which result from the pressure of the tannins
and mucilage on the epidermis or the periderm, which cause rupture of the epidermis
(Figure, 5 [A,D]).
C) The Leaf
Generally, there were no anatomical differences among the reproductive shootleaves and the non-reproductive shoot leaves, the differences were mainly
morphological.
Figure (6): A) Papillae trichome on Solanum diphyllum L. leaf . B) Multicellular
glandular trichome on Solanum diphyllum L. leaf margin (Bar = 0.05 mm.).
The leaf blade:
The lamina ofS. diphyllum L. is dorsiventral, the leaf epidermis has few sparse
trichomes of four types; unicellular papillae hairs which were rare, simple
unicellular and multicellular uniseriate 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 numerous in young
leaves. Trichomes mainly were present along the margin of the leaves and the main
vein on the abaxial laminaside (Figure, 6).
A B
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MACRO, MICRO-MORPHOLOGICAL AND BIOACTIVITY. 187
Stomata were anisocytic (unequalcelled); as stoma are surrounded by three
cells, one of them is distinctly smaller than the other two (Van Cotthem, 1970).
Stomata were very rare in the adaxial surface and more frequent on the abaxial
epidermis (Figure, 7 [A]).
Figure (7): A) Abaxial epidermal surface ofSolanum diphyllum L. leaf under light
microscope showing anisocytic stomata (Bar = 0.05 mm). B) Abaxial
epidermal surface ofSolanum diphyllum L. leaf under scanning microscope
showing the crystalloid wax and the stomata ( Bar = 70 m).
Our result agrees with Metcalfe and Chalk (1950), Ahmad (1964), and Dwelle et
al. (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 dorsiventral leaf, anisocytic stomata were confined to one
surface (Watson and Dallwitz, 1992) and simple uniseriate or multicellular
eglandular or glandular hairs (Edmonds, 1972) were the characters that have been
detected and shared with other members of family Solanaceae and genus Solanum.
The stomatal openings were ovate-rounded shaped. The electron microscope
scanning for the leaf 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 tothe presence of the rims, which give the shape of depressed stomata (Figure 7 [B]).
The mesophyll was distinguished into palisade and spongy tissues. Palisade
tissue cells were large, elongated more or less cylindrical and arranged in a single
layer. The spongy tissue cells were rounded or lack regularity and arranged loosely
in 3-5 layers, large intercellular spaces were occurred among these cells. The
palisade and spongy tissues were equally thick, with the ratio of approximately 1:1.
There were raphides crystals in the spongy tissue. In the wings of the leaf blade
there were undifferentiated small or subsidiary vascular bundles along the two
wings.
A B
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FATMA A. HAMADA, et. al188In the midrib region, the main vein is prominent, especially on the abaxial
side. On the adaxial side underneath the epidermis were present the
chlorenchymatous cells; 3-5 layers, followed by lacunar collenchyma; 3-7 layers,
and parenchyma cells of different sizes, rounded in shape, with numerous small
intercellular spaces among them; 3-5 layers. Druses crystals were detected in the
parenchymatous cells. At the middle of the midrib region there was a large
bicolateral vascular bundle, with patches of external and internal phloem (Figure, 8
[B]). Sclerenchymatous fibers were present around the vascular bundle especially in
the middle-aged and old leaves which may be considered as bundle sheath.
Some cells of the parnchymatous and spongy tissues were mucilage cellscontaining dark mucilage and druses crystals. Some other epidermal cells has tannin
sacs (Figure, 8 [C]). Tannins are polyphenolic substances that have a scattered
distribution through various plant families (e.g. Myrtaceae). The function of tannins
to some extent is still little understood, they may act as an ultraviolet light shield in
some plants where they are present in epidermal cells as very strong sunlight can
damage chloroplast so such a screen could be beneficial. In addition it can act as
herbivore deterrent as its stringent taste may protect leaves from being eaten (Cutler,
1978).
With the pressure of the tannins the periderm cracked forming lenticels likestructure. Usually the tannin cells often form connected systems and may be
associated with vascular bundles (Esau, 1977), these observation emphasizes our
suggestion that the dark components observed in the epidermal cells of Solanum
diphyllum L. may be due to tannin sacs and mucilage idioblasts (Figure, 8 [C, D]).
According to Franceschi and Homer (1980) idioblasts mucilage cells containing
crystals occur widely in the plant kingdom and have been observed in most plant
organs. These crystals may be raphides, styloids, druses, prismatics, or crystal sands
(Wang et al., 1994) in our case mucilage cells included druses crystal. Crystalcontaining cells may not differ from other parenchymatous cells, but they are more
or less specialized in form and content (Esau, 1977).
Generally Lenticels are limited interruptions in the cork and phelloderm through
which gaseous exchange with the surrounding takes place. Leaves differ from most
stems and roots in that they are almost all primary organs, although some secondary
growth can occur, as for example in the vascular supply of some gymnosperm leaves
and in the leaf bases of some monocotyledons with secondary stem growth. Large
changes do not occur in the shape or thickness of dicotyledonous leaves after
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MACRO, MICRO-MORPHOLOGICAL AND BIOACTIVITY. 189
primary growth has ceased (Cutler, 1978). Thus the presence of lenticels on
Solanum diphylllum L. leaves happened in response to something!
It was detected that most of the secreted material was discarded to the outside
only after repture of the cuticle (Schnepf, 1969), e.g. as seen in Inula. Generally, the
inner secretory tissues are characteristic of certain plants; their development may or
may not depend on external factors such as injuries, pathogens or physiological
stresses (Fahn, 1987). This secreted material is either exuded through minute pores
in the cuticle, or accumulates below the cuticle and discarded outside after cuticle
rupture (Fahn, 1988).
This was not the first time to detect lenticels associated with tannin presence, asthere were several rows of radially arranged tannin cells observed in lenticels of
Ficuss microcarpa, and the reason of its presence was not known (Kuo-Huaung and
Hung, 1995).
Figure (8): Solanum diphyllum L. leaf. A) Leaf has excretions of dark spots (Bar = 2cm).
B) T.S in the leaf shows lenticel-like structure (Bar = 0.1 mm). C) Tannin
sacs inleaf epidermis (Bar = 0.05 mm.) D) Mucilage excertion cracksleaf
epidermis (Bar = 0.05 mm).
BA
C D
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FATMA A. HAMADA, et. al190Epicuticular wax crystals were detected in many plant species on the surface of
the cuticle (Baker, 1982). These aggregates of wax serve a variety of ecological and
physiological functions. As for example it led to enlarging the exposed hydrophobic
surface, render the leaf highly unwettable (Holloway, 1970), thereby protecting the
plant from dirt (Barthlott et al. 1998) and pathogenic microorganisms (Deising et
al., 1992). Wax crystals can also help to maintain stomatal gas exchange (by
keeping water off the pores) (Brewer and Smith, 1997). Also, it serves as a
protecting and selecting barrier by reducing insect mobility (Knoll, 1914). The
presence of wax may lead to give the shape of slightly sunken stomata, the manner
of distribution of the wax particles sometimes is considered as a beneficial character.
Thomas and Barber (1974) and Cameron (1970) studied the effect of cuticular
waxes on light absorption in leaves ofEucalyptus spp. they found that differences in
characteristics caused by the amount and orientation of waxes on the leaf cuticle
caused variation in the ability of the leaves to absorb light in the 400-700nm
waveband, so the presence of wax and tannins help the plant to acclimate to the
surrounding light intensities. Generally it was observed in the field that Solanum
diphyllum L. grows widely in shade places under trees. The distribution of stomata
on the abaxial surface probably is an adaptation to water loss or light strength. This
is in agreement with Metacalf and Chalk (1950).
The leaf petiole
The cross section outline of the petiole is more or less half a circle with straight
to shallow raised adaxial surface. Epidermal cells are radially elongated covered
with thin layer of cuticle, followed by 2-3 layers of chlorencyma, lacunar
collenchyma 5-7 layers and then isodiametric parenchyma. Two small lateral ridges
or subsidiary bundles were present near the adaxial surfaces which were responsible
to feed the fimbrial veins (Figure, 9).
Figure (9): T.S in leaf petiole ofSolanum diphyllum L. (Bar = 0.05 mm).
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MACRO, MICRO-MORPHOLOGICAL AND BIOACTIVITY. 191
The rest of the vascular tissue of the petiole, which extends into the lamina as the
mid-vein, appears as a median arc-shaped (horse-shoe shaped). Bicollateral vascular
bundle, in which internal phloem is well developed than the external one, is
surrounded by scattered lignified fibers. Crystal sands were present. Tannin sacs,
mucilage cells and lenticel-like structures were present as the case in the leaf and
stem. The petiole nearly has the same tissues as the leaf and in a similar
arrangement.
Generally the collected specimens from the two different localities (Giza and
Aswan governorates) were morphologically and anatomically similar.
Analysis of certain metabolites:
The analyses of certain metabolites in the leaves, stems, and roots of Solanum
diphyllum L. showed that the total tannins and carbohydrate were found in relatively
large amounts in root and stem in comparison with other metabolites (data not
shown). However, tannins, flavonoids and saponins were found in large amounts in
leaf tissues (Figure, 10).
Beside the functions that previously recorded for the tannin sacs, in general,
polyphenols, which were proved to be high in Solanum diphyllum L. leaves extract
(Hossain et al., 2009), have a wide range of possible functions in plants; thatconcerned with basic processes of growth and development, or involved in
protection and varied according to the environmental and ecological pressures,
which they might help to minimize their hazards (Smith, 1975). Moreover,
accumulation of total phenols was detected in most xerophytic plants and could be
considered as an adaptive physiological response and safeguards against drought
stress (Abd Alla et al., 1999; Hegazi, 2000).
Figure (10): Analysis of certain metabolites in Solanum diphyllum L. different parts.
0
2
4
6
8
10
12
14
16
Leaf Stem Root
plant part
percentage%
(gm/100gmd
ry.w
t.)
Total tannins
Total carbohydrates
Total flavonoids
Total saponins
Total alkaloids
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FATMA A. HAMADA, et. al192
According to Morgan (1984) and Munns (1988) one of the main adaptivemechanisms contributing to drought resistance in plants is the ability to actively
accumulate solutes, in order to decrease the internal osmotic potential and thus
prevent or reduce an important loss of water from the stressed tissues. It was
observed by (Soliz-Guerrero et al., 2002; Moges et al., 2001; Watkinson et al.,
2003, 2006; Leatherwood, 2005; Misra and Gupta, 2006; Abdul Jaleel et al., 2007)
that the ability of the plant to tolerate drought is due to the accumulation of
compatible solutes such as polyphenols, carbohydrates, alkaloids and saponins, etc.
Knapp (2002)observed and stated that Solanum diphyllum L. is widespread indrier habitats, and has a woody tap roots, which may persist through dry or
otherwise unfavorable periods, also it was observed by Sheded et al. (2010) that
Solanum diphyllum L seeds have the ability to germinate under low osmotic
potential induced by PEG 6000, in other words it has the ability to resist drought.
All these emphasis that the tannin sacs and mucilage cells may play a role in this
process (drought resistance), and the discard of these substances via the plant stem
and the leaves epidermis may be a regulatory process; as the plant under favourable
conditions has no need for the extra stored osmolytes, this hypothesis is in need to
further investigation.
In addition, there are other morphological character such as root systems of
plants adapted to arid regions, which possess a range of unique features to survive
and reproduce in such dry conditions and poor soils. Solanum carolinense is a good
example as mentioned previously; has an extensive root system with lateral root
branches, and recorded by Bradbury and Aldrich (1957) as a drought tolerant plant.
Solanum diphyllum L. has to some extent the same root morphological character of
Solanum carolinense, and its seeds showed the ability to resist drought, moreover,
from literatures it was detected to grow in dry habitats, and it is taxonomically
related to Solanum carolinense so it could have the same ability of drought
resistance.
The relationship between plant structure and the environment in which the plant
grows was a fascinating subject to early plant anatomists, and still to be of great
interest, as the adaptations in the anatomy of plants might be an ecological benefit.
Moreover we should realize that not all adaptations are evident at the morphological
level, some are physiological (Cutler, 1978) or might be at both levels; in which the
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MACRO, MICRO-MORPHOLOGICAL AND BIOACTIVITY. 193
plant uses both of them in order to persist in the unfavorable conditions and insure
its existence.
Cytotoxicity assay:
The growth inhibition percentage was found to be increased with increasing
concentration of different crude extracts. Results are tabulated in tables (1, 2) and
graphically represented in figure (11).
The methanolic extracts were found to exhibit activity against the human colon,
breast and hepatoma cancer cell lines. The cytotoxic activity of various Solanum
diphyllum L crude extracts was clearly paralleled to the added concentration; it wasobserved that at concentration 25 g/ml the inhibition percentages of root crude
extract against HCT116, MFC7, HPEG2 carcinoma cell lines were (88%, 86.9% and
73.5%), shoot crude extract (85.5%, 83.8% and 73.3%) and fruit crude extract
(87.2%, 85.3% and 68.3%) respectively, and were less toxic for the normal human
melanocytes cell line (HFB4) in comparison with these cancer cell lines.
It was observed that the inhibition percentage begin to decline with conc. 50
g/ml and this may refer to the absence of response in the survival fraction with
increasing concentration of the crude, which may be due to saturation of the carrierprotein which transports the drug intracellularly with the drug. In addition, with
increasing time the drug is inactivated by metabolizing enzymes. Moreover, the
portions of cells that were not affected or killed by the drug proliferate normally due
to absence of the drug; as there was no longer effect of the drug in the cells (Prof.
Shouman, person. comm.).
The HCT116, MFC7, HEPG2 cells which were treated with different
concentrations of crude extracts showed antiproliferative effects under BRS assay.
The IC50 (inhibitory concentration 50%) values for root crude extract were (4.04,
5.26 and 11.5 g/ml), shoot crude extract (4.5, 9.80 and 10.6 g/ml) and fruit crude
extract (5.11, 9.07 and 10.4 g/ml) respectively, indicating that the susceptibility of
HCT116 and MFC7 cells is higher than that of HEPG2 cells. In other words, colon
and breast cancers were the most sensitive, while liver cancer is the least sensitive,
and the crude extracts were less toxic for normal cells (IC50=22 g/ml).
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FATMA A. HAMADA, et. al194Table (1): Growth inhibitory activity percentage of the three crude extracts ofSolanum
diphyllum L. against HCT116, MFC7, HEPG2 as human cancer cell lines, andHFB4 melanocytes as a normal cell line.
In terms of safety, the potent plant extracts should be less cytotoxic for the
normal human skin melanocytes cell line, than for the cancer cell lines. The resultsshowed that Solanum diphyllum L. root crude is the most potent inhibitor of
HCT116 (human colon carcinoma cells) by growth inhibition, followed by shoot
crude extract and seed crude extract respectively. Root crude extract was a potent
inhibitor against breast cancer MCF7 cells demonstrated maximum cytotoxicity (IC
50= 5.26 g/ml), and according to SRB assay results, plant crude extracts were less
toxic for normal human skin melanocytes with an IC50 of 22 g/ml.
The root crude extract was more potent than shoot and fruit crude extracts, and
the cytotoxicity value of HCT116 under the effect of root crude extract (IC 50) = 4.04
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MACRO, MICRO-MORPHOLOGICAL AND BIOACTIVITY. 195
g/ml was significantly lower than that for doxorubicin (a drug reference), which its
IC50 were (4.5 and 5 g/ml) respectively in case of HCT116 and MCF7 cancer cell
lines. Shoot crude extract (IC50 = 4.50 g/ml) was more potent in its effect than fruit
extract (IC50 = 5.11 g/ml) against HCT116 cell line.
The crude extracts were proved to have cytotoxic activity against cancer cell
lines in vitro, and were less toxic for normal human melanocytes than for the three
cancer cell lines. However the 25 g/ml concentration induced the higher inhibition,
actually it was not the best concentration, because the inhibition was not low enough
in case of the normal cell line.
Table (2): IC50 for the crude extracts and for doxorubicin as a drug reference.
Statistical analysis showed obviously that the best concentration of Solanum
diphyllum L. different crude extracts affected the carcinoma cells, and in the same
time has low toxicity on the normal cells (HFB4) was 12.5 g ml -1 (Figure 11).
Our results suggested that medium-low doses of root extract may cause
apoptosis or suppression for HCT116 and MCF7 carcinoma cells potentially,
besides shoot and seed extracts affected HCT116 and are safe as they did not affect
the normal cells severely.
According to Lacaille-Dubois (2005) several pharmacological in vitro and in
vivo researchers found various pharmacologically active compounds in many plants
e.g. Dioscorea species having cytotoxic activity, even water extracts ofDioscorea
membranacea were found to be specifically active on breast cancer cells (IC50 7.7
g/ml) with little activity on normal cells (IC50 78.4 g/ml).
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FATMA A. HAMADA, et. al196
Figure (11): Total mean surviving fraction of different crude extracts at conc. 5, 12.5
and 25 g/ml.(*) Shows no significance between different cell lines at the same concentration at
p
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MACRO, MICRO-MORPHOLOGICAL AND BIOACTIVITY. 197
hepatocellular carcinoma cells (McA-RH7777 and HEPG2). Also, It was detected
that Sukun wood crude ( Artocarpus altilis) against human breast cancer (T47D)
cells has a cytotoxic effect in a concentration dependent manner, with an extract IC 50
(6.19 g/ml) (Arung et al. 2009).
Tribulus terrestris in addition to its aphrodisiac properties, it was proved to
contain compounds that efficiently affect tumors and have been used extensively in
Indian and Chinese traditional medicine for the treatment of various urinary,
cardiovascular and gastrointestinal disorders (Wang et al., 1990; CHEMEXCIL,
1992; Anand et al., 1994). Moreover, there were compounds isolated from
fenugreek (Trigonella foenum graecum L.) induced cell death and morphological
change indicative of apoptosis in leukemia carcinoma cell line H-60 (Hibasami et
al., 2003).
Solanum aculeastrum Dunal has long been used to treat various cancers in the
Eastern Cape Province of South Africa, and compounds that has inhibitory effect on
Hela carcinoma cells were separated from it (Koduru et al., 2007). Moreover a
steroidal alkaloid compound, 3-O-(-D-Glucopyranosyl) Etioline; that exhibit a
cytotoxic effect against the Hela cells (cervical cancer cell line) was separated by El-
Sayed et al. (2009) from the methanolic extract ofSolanum diphyllum L. roots. And
It was reported that Solanum lyratum extract (SLE) inhibited cell proliferation of
gastric carcinoma SGC- 7901 cells, human hepatoma BEL-7402 cells, and A375-S2
cells in vitro and in vivo (Sun et al., 2006; Ren et al., 2006).
Doxorubicin is an anthracycline antibiotic of wide spectrum of action has been
used in healing cancer tumors since the late 1960s. It considered as a powerful drug
against cancer. The tumors are most commonly responding to doxorubicin when it is
given as a single agent or in combination with other antitumor agents. Other cancers
that are less responsive to doxorubicin are still treated with the drug because of its
overall benefits (Singal and Liskovic, 1998). Doxorubicin (DOX) is a highly
effective chemotherapeutic agent used in the treatment of solid and hematopoietic
tumors (Xin, et al., 2007).
Doxorubicin is used to treat neoplastic diseases such as acute lymphoblastic
leukemia, Wilms tumor, soft tissue and osteogenic sarcomas, Hodgkins disease,
non-Hodgkins lymphomas, Ewings sarcoma, and bronchogenic, genitourinary,
breast, and thyroid carcinoma (IARC, 1976). Doxorubicin is a highly effective
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FATMA A. HAMADA, et. al198antineoplastic drug, but its clinical use is limited by its adverse side effects on the
heart as it induced cardiotoxicity and congestive heart failure, and affected the
mature and the growing skeletons (Li et al., 2006; Van Leeuwen, et al., 2000).
Thus having a new drug that avoids the adverse side effects of Doxorubicin is
considered as a promising good merit. Our results showed that the IC50 using the
crude extracts were more potent than that of Doxorubicin, especially that of root
extract against HCT116. Moreover, a steroidal alkaloid compound, 3-O-(-D-
Glucopyranosyl) Etioline was separated by El-Sayed et al. (2009) from the
methanolic extract ofSolanum diphyllum L. root, which exhibited a cytotoxic effects
against the Hela cells (cervical cancer cell line). All these suggest the possibility of
using Solanum diphyllum L. as a potent source for anticancer compounds that could
be utilized pharmaceutically after further investigations.
Conclusion
In conclusion, the plant showed a morphological and anatomical affinity to its
family, section and genus. Root, shoot and fruit methanolic extracts of Solanum
diphyllum L exhibited cell viability decreasing in a concentration-dependent mannerand potent activity against variety of carcinoma cell Lines (breast, colon and
hepatoma cancer cells) with relatively mild effect on normal cell line, therefore it
could has a potential anti-cancer effect.
The isolation and characterization of the compound(s) responsible for the
observed activities of these extracts are currently being conducted. The plant needs
further studies in order to explore its drug therapy possibilities.
Acknowledgement
Thanks due to Kew Botanical Garden Herbarium, U.K. for providing the loaned
specimen herbarium sheet of Solanum diphylllum L. used in confirming the
identification of the plant species.
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